Notebook

DNA Transfection of HEK293T with jetOPTIMUS: 620_pBudCE4.1_MxEncA_STII-M, 320_pcDNAT7v2

Goal:

Introduction of 620_pBudCE4.1_MxEncA_STII-M, 320_pcDNAT7v2 into HEK293T cell line.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Cells were seeded in 96 well microscopic slides from ibidi at density of 5*10 3 cells per well and grown overnight. Afterwards cells were treated with jetOPTIMUS reagent mixture, including plasmid of interest and fresh DMEM. Afterwards, cells were grown at  37°C with 5% CO 2 and 90% relative humidity. We transfected twenty-one wells with 620_pBudCE4.1_MxEncA_STII-M and three wells with 320_pcDNAT7v2 as negative controls for the DOE.

Results:

The DOE results were evaluated and plotted with a platereader.

Tyrosinase Activity Assay of 670_pET_21(+)_BmTyr-Twin-Strep, 680_pET_21(+)_BmTyr(E195S+A221V), 690_pET_21(+)_BmTyr(R209S+M215E+V218M), 700_pET_21(+)_BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C), 710_pET_21(+)_cpBmTyr-Twin-Strep, 720_pET_21(+)_NBmTyr(85)-P4-Twin-Strep, 730_pET_21(+)_P3-CBmTyr(85)-Twin-Strep, 741_pET_21(+)_NBmTyr(157)-Twin-Strep, 751_pET_21(+)_CBmTyr(157)-Twin-Strep, 760_pET_21(+)_NBmTyr(201)-P4-Twin-Strep, 770_pET_21(+)_P3-CBmTyr(201)-Twin-Strep, 790_pET_21(+)_HcTyr(1-296)-TEVCS(M)-HcTyr(336-466)-TwinStrep, 810_pET_21(+)_HcTyr1(1-296)-TEVCS(M)-Twin-Strep, 820_pET_21(+)_LysTyr-Twin-Strep, 830_pET_21(+)_SavMelC1(27-118)-Twin-Strep, 840_pET_21(+)_SavMelC2-Twin-Strep, 850_pET_21(+)_SavMelC1(27-118)(Y92F)-Twin-Strep, 860_pET_21(+)_SavMelC2(I42Y)-Twin-Strep, 870_pET_21(+)_SkMelC1(33-124)-Twin-Strep, 880_pET_21(+)_SkMelC2-Twin-Strep, 890_pET_21(+)_VsTyr(37-357)-TEVCS(M)-VsTyr(371-518)-Twin-Strep, and 900_pET_21(+)_VsTyr(37-357)-TEVCS(M)-Twin-Strep

Goal:

Monitoring tyrosinase activity with high sensitivity for the constructs 670_pET_21(+)_BmTyr-Twin-Strep, 680_pET_21(+)_BmTyr(E195S+A221V), 690_pET_21(+)_BmTyr(R209S+M215E+V218M), 700_pET_21(+)_BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C), 710_pET_21(+)_cpBmTyr-Twin-Strep, 720_pET_21(+)_NBmTyr(85)-P4-Twin-Strep, 730_pET_21(+)_P3-CBmTyr(85)-Twin-Strep, 741_pET_21(+)_NBmTyr(157)-Twin-Strep, 751_pET_21(+)_CBmTyr(157)-Twin-Strep, 760_pET_21(+)_NBmTyr(201)-P4-Twin-Strep, 770_pET_21(+)_P3-CBmTyr(201)-Twin-Strep, 790_pET_21(+)_HcTyr(1-296)-TEVCS(M)-HcTyr(336-466)-TwinStrep, 810_pET_21(+)_HcTyr1(1-296)-TEVCS(M)-Twin-Strep, 820_pET_21(+)_LysTyr-Twin-Strep, 830_pET_21(+)_SavMelC1(27-118)-Twin-Strep, 840_pET_21(+)_SavMelC2-Twin-Strep, 850_pET_21(+)_SavMelC1(27-118)(Y92F)-Twin-Strep, 860_pET_21(+)_SavMelC2(I42Y)-Twin-Strep, 870_pET_21(+)_SkMelC1(33-124)-Twin-Strep, 880_pET_21(+)_SkMelC2-Twin-Strep, 890_pET_21(+)_VsTyr(37-357)-TEVCS(M)-VsTyr(371-518)-Twin-Strep, and 900_pET_21(+)_VsTyr(37-357)-TEVCS(M)-Twin-Strep.

Protocol:

Preparation of Stock Solutions

• Tyrosinase Stock Solution (2000 U/mL): Add 120 µL of Assay Buffer into the Tyrosinase Standard vial and mix well
• Tyrosinase Substrate Stock Solution (50X): Add 100 µL of purified water into the Tyrosinase Substrate vial and mix well

Preparation of Standard Solution

• Dilute the Tyrosinase stock solution (2000 U/mL) 1:5 using Assay Buffer to a final concentration of 400 U/mL to make Tyrosinase  Standard solution (T1)
• Perform 1:2 serial dilutions to produce the remaining serially diluted Tyrosinase  Standards (T2-T7)

Dilution	Tyrosinase Std	Serial Dilution Source	Assay Buffer	Concentration
T1	20	400	80	400
T2	50	From T1	50	200
T3	50	From T2	50	100
T4	50	From T3	50	50
T5	50	From T4	50	25
T6	50	From T5	50	12.5
T7	50	From T6	50	6.25

Preparation of Working Solution

• Make 1:50 dilution by adding 20 µL Tyrosinase Substrate stock solution & 20 µL Tyrosinase Enhancer into 960 µL  Assay Buffer and mix well

Assay reaction:

• add 50 µL of each standard, blank (Assay buffer) and test samples into separate wells of the 96-well plate
• Add 50 µL of Tyrosinase Substrate working solution to each well containing a standard, blank, and test sample to make the total assay volume of  100 µL/well
• Incubate at room temperature for 30 - 60 minutes

Results:

Results were evaluated by creating a standard curve and plotting the measured absorbances to the curve.

sample

A600nm

Western Blot of 330_LET_CLS-XaCS-BmTyr

Goal:

Confirm the presence of and separate expressed proteins from the construct 330_LET_CLS-XaCS-BmTyr.

Protocol:

1. Preparation of 2x 15 mm gels:

	12 %
H2O	4.2 mL
30 % Acrylamid	8.0ml
1M TrisHCl	7.4 mL
10 % SDS	200 µL
Ammoniumpersulfate (APS)	160 µL
TEMED	20 µL

For a stain-free gel, 100µl of Trichlorethanol (TCE) for 2 gels/200µl for 4 gels must be added.

All ingredients except APS and TEMED were added to a Falcon tube. The Falcontube got inverted to thoroughly mix the solution. APS and TEMED were added to initiate polymerisation just before pouring the solution into the assembled gel electrophoresis chamber. The gel will begin to polymerize immediately.By adding ~200 µL of isopropanol on top, air contact is prevented. The gel has to sit undisturbed for ~20 minutes. Once set, the isopropanol is removed and the prepared stacking gel solution is added.

Stacking gel	6 %
H2O	4.1 mL
30 % Acrylamid	1.2 mL
1M TrisHCl pH 6.8	750 µL
10 % SDS	60 µL
Ammoniumpersulfate (APS)	30 µL
TEMED	6 µL

A clean comb is inserted into the stacking gel, while avoiding air bubbles. Lastly, the gel must be kept in a vertical position at room temperature.

2. Buffer preparation

For electrophoretic separation of proteins, a 10× running buffer was prepared by dissolving 144 g of glycine, 30 g of Tris base, and 10 g of SDS in distilled water (ddH₂O), followed by adjustment of the final volume to 1 L. Prior to use, the running buffer was diluted to 1× with ddH₂O.The wet transfer buffer (Towbin buffer) used for Western blotting was prepared by dissolving 14.4 g of glycine and 3.0 g of Tris base in ddH₂O, followed by the addition of 200 mL of methanol and 3.75 mL of 10% SDS. The final volume was adjusted to 1 L with ddH₂O. As an alternative, wet transfer buffer could be prepared by diluting 100 mL of the 10× running buffer with 700 mL ddH₂O and 200 mL methanol.For semidry protein transfer, Bjerrum buffer was prepared by dissolving 2.93 g of glycine and 5.82 g of Tris base in ddH₂O, followed by the addition of 200 mL methanol and 3.75 mL of 10% SDS. The final volume was adjusted to 1 L with ddH₂O. All buffers were prepared fresh or stored at 4 °C until use.

3. SDS-PAGE electrophoresis

Protein samples were brought to room temperature (RT) before loading. Gels were inserted into the running chamber, and, in the case of precast gels, the tape at the bottom was removed to allow buffer flow. The chamber was filled with 1× running buffer, ensuring that the gel was fully submerged and that no air bubbles were present at the bottom of the cassette. Protein samples were briefly vortexed and loaded onto the gel. The electrophoresis was performed under constant voltage conditions, typically at 100 V, until sufficient protein separation was achieved. Gels were then removed and processed for protein transfer.

4. Protein transfer

For high-molecular-weight proteins, wet transfer was employed. Two Whatman papers, two sponges, and one nitrocellulose membrane per gel were equilibrated in pre-cooled Towbin transfer buffer. After equilibration, the gel was removed from the cassette and placed in the same buffer. The blotting sandwich was assembled in the following order (from anode to cathode): transparent sponge, Whatman paper, nitrocellulose membrane, gel, a second layer of Whatman paper, and black sponge. The sandwich was placed in the transfer cassette, and a cold pack was used to maintain temperature. Protein transfer was performed at constant current (400 mA) for 1.5 hours.

5. Antibody incubation

Tris-buffered saline with Tween-20 (TBS-T) was prepared by dissolving 88 g of NaCl and 24 g of Tris base in ddH₂O, adjusting the pH to 7.6, and adding 1.5 mL of Tween-20 to a final volume of 1 L. For blocking, a 5% (w/v) milk solution was prepared by dissolving 2.5 g of milk powder in 50 mL of TBS-T. Membranes were blocked in 5% milk for 1 hour at RT with gentle shaking.Primary antibodies were diluted in TBS-T and incubated with the membrane for either 1 hour at RT or overnight at 4 °C. Membranes were then washed three times for 5 minutes each with TBS-T. Secondary antibodies, also diluted in TBS-T, were incubated with the membrane for 1 hour at RT, followed by three additional washes with TBS-T.

6. Signal detection

For chemiluminescent detection, membranes were incubated briefly in enhanced chemiluminescence (ECL) solution, prepared by mixing ECL Solution 1 and ECL Solution 2 in a 1:2 ratio. Chemiluminescence was detected using the ImageLab imaging system. For imaging, a new project was opened in single-channel mode. Colorimetric detection was used for Ponceau S staining, and chemiluminescent detection (Chemi mode) was used for antibody signals. Images were acquired and saved with appropriate labels for documentation and analysis.

Results:

The samples were loaded from left to right: standard - lysate - flowthrough - wash - elution 1 - elution 2 - reference

Sanger sequencing of 630_pcDNAT7v2_iCasp9-T2A-GFP

Goal:

To confirm the nucleotide sequences of 630_pcDNAT7v2_iCasp9-T2A-GFP, Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a (partial) match between the experimental and expected sequences. In addition, the sequencing data showed (predominantly) a high quality score and a continuous read length of over 1 kb.

Sample	Barcode	Primer	Validation
630_pcDNAT7v2_iCasp9-T2A-GFP		CMV-fw	yes
630_pcDNAT7v2_iCasp9-T2A-GFP		WPRE	yes

DNA Transfection of HEK293T MESA2: 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 650_pcDNAT7v2_CD4SP-L-CD28-CS(G)-tTAEmma 320_pcDNAT7v2

Goal:

Introduction of 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 650_pcDNAT7v2_CD4SP-L-CD28-CS(G)-tTAEmma 320_pcDNAT7v2 into HEK293T cell line.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Cells were seeded in 96 well microscopic slides from ibidi at density of 5*10^3 cells per well and grown overnight. Afterwards cells were treated with jetOPTIMUS reagent mixture, including plasmid of interest and fresh DMEM. Afterwards, cells were grown at  37°C with 5% CO 2 and 90% relative humidity. We transfected in a 1:1:2:4 (Ntev : Ctev : tTA : reporter) ratio and added 200 mg / mL mCherry after 12h.

Results:

We evaluated the transfection using CellInsight™ CX7 High Content Analysis Platform from Thermo Scientific™.

DNA Transfection of HEK293T Encapsulins: 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES, 910_pcDNAT7v2_BmTyr-TEVCS(M)-MxEnc-eUnaG-STII-NES, 920_pcDNAT7v2_BmTyr-TEVCS(M)-QtEnc-eUnaG-STII-NES, 930_pcDNAT7v2_BmTyr-TEVCS(M)-TmEnc-eUnaG-STII-NES, 950_pcDNAT7v2_MxSig-BmTyr-DD-C, 970_pcDNAT7v2_TmSig-BmTyr-DD-C, 980_pcDNAT7v2_MxEnc-eUnaG-STII-NES, 990_pcDNAT7v2_QtEnc-eUnaG-STII-NES, 1000_pcDNAT7v2_TmEnc-eUnaG-STII-NES and @FG09.2

Goal:

Introduction of 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES, 910_pcDNAT7v2_BmTyr-TEVCS(M)-MxEnc-eUnaG-STII-NES, 920_pcDNAT7v2_BmTyr-TEVCS(M)-QtEnc-eUnaG-STII-NES, 930_pcDNAT7v2_BmTyr-TEVCS(M)-TmEnc-eUnaG-STII-NES, 950_pcDNAT7v2_MxSig-BmTyr-DD-C, 970_pcDNAT7v2_TmSig-BmTyr-DD-C, 980_pcDNAT7v2_MxEnc-eUnaG-STII-NES, 990_pcDNAT7v2_QtEnc-eUnaG-STII-NES, 1000_pcDNAT7v2_TmEnc-eUnaG-STII-NES and @FG09.2 into HEK293T cell line.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Cells were seeded in 24 well microscopic slides from ibidi at density of 5*10 3 cells per well and grown overnight. Afterwards cells were treated with jetOPTIMUS reagent mixture, including plasmid of interest and fresh DMEM. Afterwards, cells were grown at  37°C with 5% CO 2 and 90% relative humidity.

Results:

The results were evaluated in downstream experiments.

DNA Transfection of HEK293T soluble MESA: 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 650_pcDNAT7v2_CD4SP-L-CD28-CS(G)-tTAEmma>

2025-09-29

Anna Tichá

Goal:

Introduction of 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 650_pcDNAT7v2_CD4SP-L-CD28-CS(G)-tTAEmma>

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Cells were seeded in 96 well microscopic slides from ibidi at density of 5*10 3 cells per well and grown for 6-8 h. Afterwards cells were treated with jetOPTIMUS reagent mixture, including plasmid of interest and fresh DMEM. Afterwards, cells were grown at  37°C with 5% CO2 and 90% relative humidity. We transfected in a 1:1:4 (receptor : tTA : reporter) ratio and added 0.1 µM of rapamycin 12 h post transfection.

Results:

We evaluated the transfection using CellInsight™ CX7 High Content Analysis Platform from Thermo Scientific™.

Seeding of Nunc MicroWell 96-Well plates

Goal:

Seeding Nunc™ MicroWell™ 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate (Thermo Scientific™) for further confocal analysis of MESA transfected into HEK293T cells with \(1 \times 10^4\) HEK293T.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet. Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 37 °C for 5 minutes. Upon confirming detachment using a light phase microscope, cells were rescued using 3 ml pre-warmed complete Dulbecco's modified Eagle medium F12 (DMEM) and transferred to a 15 ml tube. Cell suspension was centrifuged at 25 °C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using a Pasteur pipette and the cell pellet was resuspended in 6 ml pre-warmed DMEM. Each well of a Nunc™ 96-well plate was inoculated with 100 µl of cell suspension at \(1 \times 10^4\) cells per well.  Cells were further grown at 37 °C with 5% CO₂ and 90% relative humidity.

Results:

Countess™ II automated cell counter results:

Chamber	Cell count (\(\times 10^6\))	Live cells (\(\times 10^6\))	Dead cells (\(\times 10^3\))
A	2.3 x10⁶	98%	2%
B	2.6 x10⁶	95%	5%

Sanger sequencing of 720_pET_21(+)_NBmTyr(85)-P4-Twin-Strep, 730_pET_21(+)_P3-CBmTyr(85)-Twin-Strep, 760_pET_21(+)_NBmTyr(201)-P4-Twin-Strep, and 770_pET_21(+)_P3-CBmTyr(201)-Twin-Strep

Goal:

To confirm the nucleotide sequences of …, Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a (partial) match between the experimental and expected sequences. In addition, the sequencing data showed (predominantly) a high quality score and a continuous read length of over 1 kb.

Sample	Barcode	Primer	Validation
720_pET_21(+)_NBmTyr(85)-P4-Twin-Strep		T7	yes
730_pET_21(+)_P3-CBmTyr(85)-Twin-Strep		T7	no
760_pET_21(+)_NBmTyr(201)-P4-Twin-Strep		T7	yes
770_pET_21(+)_P3-CBmTyr(201)-Twin-Strep		T7	yes

Sanger sequencing of 950_pcDNAT7v2_MxSig-BmTyr-DD-C, 960_pcDNAT7v2_QtSig-BmTyr-DD-C, and 970_pcDNAT7v2_TmSig-BmTyr-DD-C

Goal:

To confirm the nucleotide sequences of 960_pcDNAT7v2_QtSig-BmTyr-DD-C, 970_pcDNAT7v2_TmSig-BmTyr-DD-C, and 970_pcDNAT7v2_TmSig-BmTyr-DD-C, Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a (partial) match between the experimental and expected sequences. In addition, the sequencing data showed (predominantly) a high quality score and a continuous read length of over 1 kb.

Sample	Barcode	Primer	Validation
960_pcDNAT7v2_QtSig-BmTyr-DD-C		CMV-fw	yes
960_pcDNAT7v2_QtSig-BmTyr-DD-C		WPRE	yes
970_pcDNAT7v2_TmSig-BmTyr-DD-C		CMV-fw	no
970_pcDNAT7v2_TmSig-BmTyr-DD-C		WPRE	no
980_pcDNAT7v2_MxEnc-eUnaG-STII-NES		CMV-fw	yes
980_pcDNAT7v2_MxEnc-eUnaG-STII-NES		WPRE	yes

Sanger sequencing of 900_pET_21(+)_VsTyr(37-357)-TEVCS(M)-Twin-Strep and 940_pcDNAT7v2_TyrBm-DD-C

Goal:

To confirm the nucleotide sequences of 900_pET_21(+)_VsTyr(37-357)-TEVCS(M)-Twin-Strep and 940_pcDNAT7v2_TyrBm-DD-C, Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a (partial) match between the experimental and expected sequences. In addition, the sequencing data showed (predominantly) a high quality score and a continuous read length of over 1 kb.

Sample	Barcode	Primer	Validation
900_pET_21(+)_VsTyr(37-357)-TEVCS(M)-Twin-Strep		T7	yes
900_pET_21(+)_VsTyr(37-357)-TEVCS(M)-Twin-Strep		T7-Term	yes
940_pcDNAT7v2_TyrBm-DD-C		Cmv-fw	yes
940_pcDNAT7v2_TyrBm-DD-C		WPRE	yes

Sanger sequencing of 690_pET_21(+)_BmTyr(R209S+M215E+V218M), 790_pET_21(+)_HcTyr(1-296)-TEVCS(M)-HcTyr(336-466)-TwinStrep, 810_pET_21(+)_HcTyr1(1-296)-TEVCS(M)-Twin-Strep, 820_pET_21(+)_LysTyr-Twin-Strep, 830_pET_21(+)_SavMelC1(27-118)-Twin-Strep, 840_pET_21(+)_SavMelC2-Twin-Strep, 860_pET_21(+)_SavMelC2(I42Y)-Twin-Strep, 880_pET_21(+)_SkMelC2-Twin-Strep, 890_pET_21(+)_VsTyr(37-357)-TEVCS(M)-VsTyr(371-518)-Twin-Strep

Goal:

To confirm the nucleotide sequences of 690_pET_21(+)_BmTyr(R209S+M215E+V218M), 790_pET_21(+)_HcTyr(1-296)-TEVCS(M)-HcTyr(336-466)-TwinStrep, 810_pET_21(+)_HcTyr1(1-296)-TEVCS(M)-Twin-Strep, 820_pET_21(+)_LysTyr-Twin-Strep, 830_pET_21(+)_SavMelC1(27-118)-Twin-Strep, 840_pET_21(+)_SavMelC2-Twin-Strep, 860_pET_21(+)_SavMelC2(I42Y)-Twin-Strep, 880_pET_21(+)_SkMelC2-Twin-Strep, and 890_pET_21(+)_VsTyr(37-357)-TEVCS(M)-VsTyr(371-518)-Twin-Strep, Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a (partial) match between the experimental and expected sequences. In addition, the sequencing data showed (predominantly) a high quality score and a continuous read length of over 1 kb.

Sample	Barcode	Primer	Validation
690_pET_21(+)_BmTyr(R209S+M215E+V218M)		T7	yes
790_pET_21(+)_HcTyr(1-296)-TEVCS(M)-HcTyr(336-466)-TwinStrep		T7	yes
810_pET_21(+)_HcTyr1(1-296)-TEVCS(M)-Twin-Strep		T7	yes
820_pET_21(+)_LysTyr-Twin-Strep		T7	yes
830_pET_21(+)_SavMelC1(27-118)-Twin-Strep		T7	yes
840_pET_21(+)_SavMelC2-Twin-Strep		T7	yes
860_pET_21(+)_SavMelC2(I42Y)-Twin-Strep		T7	yes
880_pET_21(+)_SkMelC2-Twin-Strep		T7	yes
890_pET_21(+)_VsTyr(37-357)-TEVCS(M)-VsTyr(371-518)-Twin-Strep		T7	yes

Sanger sequencing of 670_pET_21(+)_BmTyr-Twin-Strep, 680_pET_21(+)_BmTyr(E195S+A221V), 690_pET_21(+)_BmTyr(R209S+M215E+V218M), 710_pET_21(+)_cpBmTyr-Twin-Strep, 721_pET_21(+)_NBmTyr(85)-Twin-Strep, 731_pET_21(+)_CBmTyr(85)-Twin-Strep, 741_pET_21(+)_NBmTyr(157)-Twin-Strep, 751_pET_21(+)_CBmTyr(157)-Twin-Strep, and 761_pET_21(+)_NBmTyr(201)-Twin-Strep, 771_pET_21(+)_CBmTyr(201)-Twin-Strep, 790_pET_21(+)_HcTyr(1-296)-TEVCS(M)-HcTyr(336-466)-TwinStrep, 810_pET_21(+)_HcTyr1(1-296)-TEVCS(M)-Twin-Strep, 820_pET_21(+)_LysTyr-Twin-Strep, 830_pET_21(+)_SavMelC1(27-118)-Twin-Strep, 850_pET_21(+)_SavMelC1(27-118)(Y92F)-Twin-Strep, 870_pET_21(+)_SkMelC1(33-124)-Twin-Strep, 880_pET_21(+)_SkMelC2-Twin-Strep, 890_pET_21(+)_VsTyr(37-357)-TEVCS(M)-VsTyr(371-518)-Twin-Strep, and 900_pET_21(+)_VsTyr(37-357)-TEVCS(M)-Twin-Strep

Goal:

To confirm the nucleotide sequences of 670_pET_21(+)_BmTyr-Twin-Strep, 680_pET_21(+)_BmTyr(E195S+A221V), 690_pET_21(+)_BmTyr(R209S+M215E+V218M), 710_pET_21(+)_cpBmTyr-Twin-Strep, 721_pET_21(+)_NBmTyr(85)-Twin-Strep, 731_pET_21(+)_CBmTyr(85)-Twin-Strep, 741_pET_21(+)_NBmTyr(157)-Twin-Strep, 751_pET_21(+)_CBmTyr(157)-Twin-Strep, and 761_pET_21(+)_NBmTyr(201)-Twin-Strep, 771_pET_21(+)_CBmTyr(201)-Twin-Strep, 790_pET_21(+)_HcTyr(1-296)-TEVCS(M)-HcTyr(336-466)-TwinStrep, 810_pET_21(+)_HcTyr1(1-296)-TEVCS(M)-Twin-Strep, 820_pET_21(+)_LysTyr-Twin-Strep, 830_pET_21(+)_SavMelC1(27-118)-Twin-Strep, 850_pET_21(+)_SavMelC1(27-118)(Y92F)-Twin-Strep, 870_pET_21(+)_SkMelC1(33-124)-Twin-Strep, 880_pET_21(+)_SkMelC2-Twin-Strep, 890_pET_21(+)_VsTyr(37-357)-TEVCS(M)-VsTyr(371-518)-Twin-Strep, and 900_pET_21(+)_VsTyr(37-357)-TEVCS(M)-Twin-Strep, Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a (partial) match between the experimental and expected sequences. In addition, the sequencing data showed (predominantly) a high quality score and a continuous read length of over 1 kb.

Sample	Barcode	Primer	Validation
670_pET_21(+)_BmTyr-Twin-Strep		T7	yes
680_pET_21(+)_BmTyr(E195S+A221V)		T7	yes
690_pET_21(+)_BmTyr(R209S+M215E+V218M)		T7	yes
710_pET_21(+)_cpBmTyr-Twin-Strep		T7	no
721_pET_21(+)_NBmTyr(85)-Twin-Strep		T7	yes
731_pET_21(+)_CBmTyr(85)-Twin-Strep		T7	yes
741_pET_21(+)_NBmTyr(157)-Twin-Strep		T7	yes
751_pET_21(+)_CBmTyr(157)-Twin-Strep		T7	yes
761_pET_21(+)_NBmTyr(201)-Twin-Strep		T7	yes
771_pET_21(+)_CBmTyr(201)-Twin-Strep		T7	yes
790_pET_21(+)_HcTyr(1-296)-TEVCS(M)-HcTyr(336-466)-TwinStrep		T7	no
810_pET_21(+)_HcTyr1(1-296)-TEVCS(M)-Twin-Strep		T7	no
820_pET_21(+)_LysTyr-Twin-Strep		T7	no
830_pET_21(+)_SavMelC1(27-118)-Twin-Strep		T7	no
850_pET_21(+)_SavMelC1(27-118)(Y92F)-Twin-Strep		T7	no
870_pET_21(+)_SkMelC1(33-124)-Twin-Strep		T7	yes
880_pET_21(+)_SkMelC2-Twin-Strep		T7	yes
890_pET_21(+)_VsTyr(37-357)-TEVCS(M)-VsTyr(371-518)-Twin-Strep		T7	no
900_pET_21(+)_VsTyr(37-357)-TEVCS(M)-Twin-Strep		T7	no

Sanger sequencing of 980_pcDNAT7v2_MxEnc-eUnaG-STII-NES, 990_pcDNAT7v2_QtEnc-eUnaG-STII-NES, and 1000_pcDNAT7v2_TmEnc-eUnaG-STII-NES

Goal:

To confirm the nucleotide sequences of 980_pcDNAT7v2_MxEnc-eUnaG-STII-NES, 990_pcDNAT7v2_QtEnc-eUnaG-STII-NES, and 1000_pcDNAT7v2_TmEnc-eUnaG-STII-NES, Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a (partial) match between the experimental and expected sequences. In addition, the sequencing data showed (predominantly) a high quality score and a continuous read length of over 1 kb.

Sample	Barcode	Primer	Validation
980_pcDNAT7v2_MxEnc-eUnaG-STII-NES		CMV-fw	yes
980_pcDNAT7v2_MxEnc-eUnaG-STII-NES		WPRE	yes
990_pcDNAT7v2_QtEnc-eUnaG-STII-NES		CMV-fw	yes
990_pcDNAT7v2_QtEnc-eUnaG-STII-NES		WPRE	yes
1000_pcDNAT7v2_TmEnc-eUnaG-STII-NES		CMV-fw	yes
1000_pcDNAT7v2_TmEnc-eUnaG-STII-NES		WPRE	yes
650_pcDNAT7v2_CD4SP-L-CD28-CS(G)-tTAEmma>		CMV-fw	yes
650_pcDNAT7v2_CD4SP-L-CD28-CS(G)-tTAEmma>		WPRE	yes

Sanger sequencing of 910_pcDNAT7v2_BmTyr-TEVCS(M)-MxEnc-eUnaG-STII-NES, 920_pcDNAT7v2_BmTyr-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 930_pcDNAT7v2_BmTyr-TEVCS(M)-TmEnc-eUnaG-STII-NES, and 660_pET_21(+)_Twin-Strep

Goal:

To confirm the nucleotide sequences of 910_pcDNAT7v2_BmTyr-TEVCS(M)-MxEnc-eUnaG-STII-NES, 920_pcDNAT7v2_BmTyr-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 930_pcDNAT7v2_BmTyr-TEVCS(M)-TmEnc-eUnaG-STII-NES, Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a (partial) match between the experimental and expected sequences. In addition, the sequencing data showed (predominantly) a high quality score and a continuous read length of over 1 kb.

Sample	Barcode	Primer	Validation
910		CMV-fw	yes
910		WPRE	yes
920		CMV-fw	yes
920		WPRE	yes
930		CMV-fw	yes
930		WPRE	yes
660		T7	yes

Splitting of HEK293T cell line into T25 flask - p21

Goal:

Splitting HEK293t cell line from "HEK VI-X" flask  to the next passage number of p21.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Culturing media was decanted from the 25 cm 2 culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos's modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 25 cm 2 flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO 2 and 90% relative humidity.

Results:

Splitting HEK293t cell line from "HEK VI-X" flask  to the next passage number of p21.

Splitting of HEK293T cell line into T25 flask - p20

Goal:

Splitting HEK293t cell line from "HEK VI-X" flask  to the next passage number of p20.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Culturing media was decanted from the 25 cm 2 culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos's modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 25 cm 2 flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO 2 and 90% relative humidity.

Results:

Splitting HEK293t cell line from "HEK VI-X" flask  to the next passage number of p20.

Splitting of HEK293T cell line into T25 flask - p19

Goal:

Splitting HEK293t cell line from "HEK VI-X" flask  to the next passage number of p19.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Culturing media was decanted from the 25 cm 2 culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos's modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 25 cm 2 flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO 2 and 90% relative humidity.

Results:

Splitting HEK293t cell line from "HEK VI-X" flask  to the next passage number of p19.

Splitting of HEK293T cell line into T25 flask - p18

Goal:

Splitting HEK293t cell line from "HEK VI-X" flask  to the next passage number of p18.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Culturing media was decanted from the 25 cm 2 culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos's modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 25 cm 2 flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO 2 and 90% relative humidity.

Results:

Splitting HEK293t cell line from "HEK VI-X" flask  to the next passage number of p18.

Splitting of HEK293T cell line into T25 flask - p17

Goal:

Splitting HEK293t cell line from "HEK VI-X" flask  to the next passage number of p17.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Culturing media was decanted from the 25 cm 2 culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos's modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 25 cm 2 flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO 2 and 90% relative humidity.

Results:

Splitting HEK293t cell line from "HEK VI-X" flask  to the next passage number of p17.

Splitting of HEK293T cell line into T25 flask - p16

Goal:

Splitting HEK293t cell line from "HEK VI-X" flask  to the next passage number of p16.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Culturing media was decanted from the 25 cm 2 culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos's modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 25 cm 2 flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO 2 and 90% relative humidity.

Results:

HEK293t cell line from "HEK VI-X" flask was split to passage number p16.

Splitting of HEK293T cell line into T25 flask - p15

Goal:

Splitting HEK293t cell line from "HEK VI-X" flask  to the next passage number of p15.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Culturing media was decanted from the 25 cm 2 culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos's modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 25 cm 2 flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO 2 and 90% relative humidity.

Results:

HEK293t cell line from "HEK VI-X" flask was split to passage number p15.

Splitting of HEK293T cell line into T25 flask - p14

Goal:

Splitting HEK293t cell line from "HEK VI-X" flask  to the next passage number of p14.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Culturing media was decanted from the 25 cm 2 culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos's modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 25 cm 2 flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO 2 and 90% relative humidity.

Results:

HEK293t cell line from "HEK VI-X" flask was split to passage number p14.

SDS Page: 670_pET_21(+)_BmTyr-Twin-Strep, 680_pET_21(+)_BmTyr(E195S+A221V), 690_pET_21(+)_BmTyr(R209S+M215E+V218M), 720_pET_21(+)_NBmTyr(85)-P4-Twin-Strep, 730_pET_21(+)_P3-CBmTyr(85)-Twin-Strep, 741_pET_21(+)_NBmTyr(157)-Twin-Strep, 751_pET_21(+)_CBmTyr(157)-Twin-Strep, 760_pET_21(+)_NBmTyr(201)-P4-Twin-Strep, 770_pET_21(+)_P3-CBmTyr(201)-Twin-Strep, 790_pET_21(+)_HcTyr(1-296)-TEVCS(M)-HcTyr(336-466)-TwinStrep, 810_pET_21(+)_HcTyr1(1-296)-TEVCS(M)-Twin-Strep, 820_pET_21(+)_LysTyr-Twin-Strep, 830_pET_21(+)_SavMelC1(27-118)-Twin-Strep, 840_pET_21(+)_SavMelC2-Twin-Strep, 850_pET_21(+)_SavMelC1(27-118)(Y92F)-Twin-Strep, 860_pET_21(+)_SavMelC2(I42Y)-Twin-Strep, 870_pET_21(+)_SkMelC1(33-124)-Twin-Strep, 880_pET_21(+)_SkMelC2-Twin-Strep, 890_pET_21(+)_VsTyr(37-357)-TEVCS(M)-VsTyr(371-518)-Twin-Strep, 900_pET_21(+)_VsTyr(37-357)-TEVCS(M)-Twin-Strep

Goal:

Using Novex™ Tris-Glycine Gels precast polyacrylamide gels for optimal separation and resolution of a broad range of our Tyrosinase cell lysate (670_pET_21(+)_BmTyr-Twin-Strep, 680_pET_21(+)_BmTyr(E195S+A221V), 690_pET_21(+)_BmTyr(R209S+M215E+V218M), 720_pET_21(+)_NBmTyr(85)-P4-Twin-Strep, 730_pET_21(+)_P3-CBmTyr(85)-Twin-Strep, 741_pET_21(+)_NBmTyr(157)-Twin-Strep, 751_pET_21(+)_CBmTyr(157)-Twin-Strep, 760_pET_21(+)_NBmTyr(201)-P4-Twin-Strep, 770_pET_21(+)_P3-CBmTyr(201)-Twin-Strep, 790_pET_21(+)_HcTyr(1-296)-TEVCS(M)-HcTyr(336-466)-TwinStrep, 810_pET_21(+)_HcTyr1(1-296)-TEVCS(M)-Twin-Strep, 820_pET_21(+)_LysTyr-Twin-Strep, 830_pET_21(+)_SavMelC1(27-118)-Twin-Strep, 840_pET_21(+)_SavMelC2-Twin-Strep, 850_pET_21(+)_SavMelC1(27-118)(Y92F)-Twin-Strep, 860_pET_21(+)_SavMelC2(I42Y)-Twin-Strep, 870_pET_21(+)_SkMelC1(33-124)-Twin-Strep, 880_pET_21(+)_SkMelC2-Twin-Strep, 890_pET_21(+)_VsTyr(37-357)-TEVCS(M)-VsTyr(371-518)-Twin-Strep, 900_pET_21(+)_VsTyr(37-357)-TEVCS(M)-Twin-Strep) under denaturing gel electrophoresis conditions.

Protocol:

Prepare 1X Sample Buffer for dilutions of samples if needed. Volumes are provided for a 40-μL sample size. Scale volumes proportionally for larger sample sizes. Heat denaturing samples at 85°C for 2 minutes. Do not heat native samples. Add 100 mL of 10X Tris-Glycine SDS Running Buffer to 900 mL of deionized water to prepare 1X SDS Running Buffer. Prepare gel by removing the comb, and rinsing the gell wells three times using 1X Running Buffer, followed by removing the white tape near the bottom of the gel cassettes  and placing the gels in the gel tank. Load the buffers by filling the chambers with the appropriate 1X running buffer. Next, load the appropriate volume of your samples in the appropriate wells and load your protein ladder in the appropriate well. Run the gel for 40 min at 200 V and stained in Coomassie overnight.

Result:

The gel appeared to be empty with exception of 720_pET_21(+)_NBmTyr(85)-P4-Twin-Strep, 730_pET_21(+)_P3-CBmTyr(85)-Twin-Strep, 741_pET_21(+)_NBmTyr(157)-Twin-Strep, 790_pET_21(+)_HcTyr(1-296)-TEVCS(M)-HcTyr(336-466)-TwinStrep and 900_pET_21(+)_VsTyr(37-357)-TEVCS(M)-Twin-Strep.

Sanger sequencing of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

To confirm the nucleotide sequences of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES, Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a (partial) match between the experimental and expected sequences. In addition, the sequencing data showed (predominantly) a high quality score and a continuous read length of over 1 kb.

Sample	Barcode	Primer	Validation
600		CMV-fw	yes
600		WPRE	yes
610		CMV-fw	yes
610		WPRE	yes

BCA protein kit

Goal:

Determine the protein concentration of

Preparation of BSA standard

	Vial	Volume of dilutent µL	Volume of BSA stock	Final BSA concentration
1	A	0	300 of stock	2000 µg/mL
2	B	125	375 of stock	1500 µg/mL
3	C	325	325of stock	1000 µg/mL
4	D	175	175 of B	750 µg/mL
5	E	325	325 of C	500 µg/mL
6	F	325	325 of E	250 µg/mL
7	G	325	325 of F	125 µg/mL
8	H	400	100 of G	25 µg/mL
9	I	400	0	0 µg/mL

>

Preparation of BCA working reagent:

• The total volume of WR required for the assay: (# standards + # unknowns) × (# replicates) × (volume of WR per sample) = total volume WR requiredNote:  200 µL of WR reagent is required for each sample in the microplate procedure

	# of standards	# samples	# of replicates	WR required	+pipetting buffer
1	9	31	2	16 mL	17.6

• Prepare WR by mixing 50 parts of BCA reagent A with 1 part of BCA reagent B (50:1, Reagent A:B)
• 17.25 mL BCA reagent A
• 0.35 mL BCA reagent B

Protocol:

1. Pipette 25 µL of each standard or sample replicate into microplate well
2. Add 200 µL of WR to each well & mix plate throughly on a plate shaker for 30 seconds with Multichannel pipette
3. Cover plate & incubate at 37 °C for 30 minutes in incubator
4. Equilibrate plate to room temperature. Measure absorbance at 562 nm on plate reader
5. Subtract the average 562 nm absorbance measurement of the blank standard replicates from the 562 nm measurements of all other individual standard and unknown sample replicate
6. Prepare a standard curve by plotting the average blank–corrected 562 nm measurement for each BSA standard vs. its concentration in µg/mL. Use the standard curve to determine the protein concentration of each unknown sample

Double-restriction digest 200_pcDNAT7v1 with Noc1 & Sac1 for downstream cloning of constructs: 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4.

Goal:

Restriction digest of the Backbone (200) from tubes 1 and 2 (frigde) with Noc1 und Sac1 enzyme, for downstream cloning of constructs: 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4.

Protocol:

Restriction digest was carried out using the Noc1 and Sac1 enzyme(s) (New England Biolabs)  according to the manufacturers protocol. Template DNA was supplied in amount of 1 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL each of Noc1 and Sac1 in a PCR tube. Samples from both backbone tubes were used, so 2 PCR Tubes were used. The samples were mixed by pipetting, and incubated at 37°C for 5 hour.

Component	Volume
Noc1, Sac1	10 U (1 μL)
rCutSmart buffer	5 μL
Backbone (200)	1 μg
Nuclease-free Water	To 50 μL

Added 5 μL of QuickCIP (recommended 1 μL / 20 μL by manufacturer), but since using 6kb Vector, we used 5 μL at 37 C for 20 minutes.

The reaction was then stopped using heat inactivation (80°C, 20 minutes).

Cleanup using Monarch PCR and DNA purification kit.

Results:

The efficiency of restriction digest was evaluated by downstream experiments.

E. coli colony picking rom selection plate containing constructs 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E) , 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP , 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP , 280_pcDNAT7v1_RS20-cpEFGP-CaM , 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) , and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

Colony picking of individual DH5α E. coli bacterial colonies from selection plate containing constructs 210, 250, 260, 280, 290, and 310 was performed for the replication and expression of the desired genetic material.

Protocol:

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with ...) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results:

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Restriction digest of 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4.

Goal:

Restriction digest of the backbone 200.1 with SacI-HF & NcoI-HF enzyme, for downstream cloning of the constructs 210, 250, 260, 280, 290, and 310.

Protocol:

Restriction digest was carried out using the SacI-HF & NcoI-HF enzyme(s) ( ...,   New England Biolabs)  according to the manufacturers protocol. Template DNA was supplied in amount of 1 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of ... in a PCR tube. The sample was mixed by pipetting, and incubated at 37°C for 1 hour. The reaction was then stopped using heat inactivation 80°C, 20 minutes).

Component	Volume
Restriction enzyme	10 U (1 μL)
rCutSmart buffer	5 μL
DNA	1 μg (1 µL)
Nuclease-free Water	To 50 μL

The reaction was then stored at -20°C or immediately used for downstream experiments .

Results:

The efficiency of restriction digest was evaluated by measuring the concentration.

• BB digest_1
• concentration: 1.59 ng/µL
• ratio 260/280: 0.79
• ratio 260/230: -0.13
• BB digest_
• concentration: 0.72 ng/µL
• ratio 260/280: 1.28
• ratio 260/230: -0.06

DNA dephosphorylation

Goal:

Dephosphorylation of 5′ ends of DNA  was performed for to prevent re-ligation of the digested vector.

Protocol:

Dephosphorylation of 5′ ends of DNA was carried out using Quick CIP ( New England Biolabs) according to the manufacturers protocol. Template DNA was supplied in amount of 10 pmol of DNA ends (about 1 μg of a 3 kb plasmid) and combined with 10 μL of Quick CIP in the PCR-tube of the digest. The sample was mixed by pipetting, and incubated at 37°C for 1 hour. The reaction was then stopped using heat inactivation in 80°C for 2 minutes.

Component	Volume
DNA	1 pmol of DNA ends
Quick CIP	10 μL

The reaction was stored at -20°C or immediately used for downstream experiments .

Results:

The efficiency of dephosphorylation of DNA ends was evaluated by downstream experiments.

Sanger sequencing of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP

Goal:

To confirm the nucleotide sequences of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a (partial) match between the experimental and expected sequences. In addition, the sequencing data showed (predominantly) a high quality score and a continuous read length of over 1 kb.

Bacterial (Re-)Transformation 200_pcDNAT7v1

GoalIntegrate 210 backbone from 2024 into bacteria on a plate and in liquid culture.

Protocol

1. Get a box of ice.
2. Get competent E. coli cells from -80°C freezer & let it thaw in your hand.
3. Gently mix & pipette 50 µL of cells into a fresh Eppendorf tube which is placed on ice.
4. Add 5 µL containing 1 pg-100 ng of backbone plasmid DNA to the cell culture. Carefully flick the tube 4-5 times to mix the cells & DNA. Do not vortex!
5. Place the mixture on ice for 10 min. Do not mix! Warm up heat block in the meantime and get agar plate. Plate can be placed in the incubator to dry them for a bit.
6. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix!
7. Place the tube on ice for 3 minutes. Do not mix!
8. Pipette 200 µL of room temperature SOC into the mixture to recover the cells.
9. Immediately spread 50-100 µL onto a selection plate and spread the liquid evenly.
10. Incubate the plate overnight at 37°C with the lid placed downwards.
11. Pour 200 mL LB + Carb in a flask and pipette 200-500 µL of the transformation into the medium.
12. Incubate the flask in the shaker overnight at 37°C.

ResultPlate: Small colonies grew overnight. Plate was sealed and placed in the cooler.Flask: After 1 day, the OD maintained 0 and no culture could be detected. After 2 days, the cell density has increased and the medium appeared cloudy.

Agarose Gel Electrophoresis for 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4, 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Protocol:

Agarose gel electrophoresis was performed using a 1,0% w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 2 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5–10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right: Ladder - 171 - 172 - 173 - 153 - 154 - 155 - Ladder.Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

PCR (Platinum™ SuperFi™ II): 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4, 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from @171, @172, @173, @153, @154, and @155 for constructs 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4, 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES with high fidelity and efficiency.

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

label	gBlock	Construct	Primer	length	extension time
1	171	590	146_F_gBlock & 147_R_gBlock	1.7 kb	1 min
2	172	600	146_F_gBlock & 147_R_gBlock	1.7 kb	1 min
3	173	610	146_F_gBlock & 147_R_gBlock	1.67	1 min
4	153	280	282_R_CaM-NcoI_pcDNA & 281_F_SacI-RS20_T7p	1.3	45 sec
5	154	290	281_F_SacI-RS20_T7p & 292_R_CTEVp-Stop-NcoI_T7tt	1.5	55 sec
6	155	310	281_F_SacI-RS20_T7p & 312_R_P4-Stop-NcoI_T7tt	0.7	30 sec

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA	10 ng	1 μL
DMSO (only for 590, 600, 610)	2.5 % (v/v)	0.5 µL
Nuclease-free water	–	6.5 μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	10 sec	35
Annealing	58°C	20 sec
Extension	72°C	30 seconds/kb
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

Agarose Gel Electrophoresis of 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES redo

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Protocol:

Agarose gel electrophoresis was performed using a 1,0% w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5–10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right: L-590-600-610 Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

PCR Clean-up (NEB) of 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES & 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES redo

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of 2x 10 μL of preheated TE buffer to the column. Following 5 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES	89.43	1.91	2.10
600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES	70.71	1.99	2.55
610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES	98.59	1.93	2.48

PCR (Platinum™ SuperFi™ II) of 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from @171, @172, and @173 for constructs 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES with high fidelity and efficiency.

gBlock	Construct	Primer	length	extension time
171	590	146_F_gBlock & 147_R_gBlock	1.7 kb	1 min
172	600	146_F_gBlock & 147_R_gBlock	1.7 kb	1 min
173	610	146_F_gBlock & 147_R_gBlock	1.67	1 min

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA	10 ng	1 μL
DMSO	2.5 % (v/v)	0.5 µL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	5–10 sec	35
Annealing	58°C	20 sec
Extension	72°C	30 seconds/kb
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

E. coli colony picking 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP .5, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP .3, 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP .5, 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP.4

Goal

Colony picking of individual DH10β E. coli (Cat. No. C3019, New England Biolabs) bacterial colonies transformed with 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) .5, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP .3, 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP .5, 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP .4 from selection plates containing carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plates. Each colony was transferred to 5 mL of fresh culture medium (LB medium with 100 μg/mL carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm (CERTOMAT BS-1 Incubator Shaker, Sartorius) for further growth and amplification of the selected cells. The agar plates were sealed to prevent contamination and stored at 4 °C.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial cultures after overnight incubation at 37 °C.

TXTL of 330_LET_CLS-XaCS-BmTyr - 580_pAAVS1_CAGp-HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K)_IRES-BSD Copy

Goal:

Cell-free transcription-translation (TXTL) reaction was performed to 330_LET_CLS-XaCS-BmTyr, 340_LET_CLS-XaCS-BmTyr(E195S+A221V), 330_LET_CLS-XaCS-BmTyr, 340_LET_CLS-XaCS-BmTyr(E195S+A221V), 350_LET_CLS-XaCS-BmTyr(R209S+M215E+V218M), 360_LET_CLS-XaCS-BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C), 370_LET_CLS-XaCS-cpBmTyr(201), 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201), 440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466), 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M), 460_LET_CLS-XaCS-LsTyr, 470_LET_CLS-XaCS-SkMelC1(33-124), 480_LET_CLS-XaCS-SkMelC2, 490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518), 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M), 510_LET_CLS-XaCS-SavMelC1(27-118), 520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F), 530_LET_CLS-XaCS-SavMelC2, 540_LET_CLS-XaCS-SavMelC2(I42Y), 550_pCas9_sgAAVS1_Cas9-HA-2xNLS-i53, 560_pAAVS1_CAGp-IL7Ra-IRES-BSD, 570_pAAVS1_CAGp-HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)_IRES-BSD, 580_pAAVS1_CAGp-HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K)_IRES-BSD Copy (e.g. in vitro-express tyrosinase for enzyme activity testing).

Protocol:

TXTL was performed using proprietary E.coli cell extract (Invitris). For this, 7 μL of Buffer B (Invitris) was mixed with 0.75 μL of dNTPs, 0.3 μL of GamS and 5 μL of cell extract. The mixture was then incubated on ice for 5-10 mins, following by addition of 0.9 μL of PEG-8000. The DNA was added at the concentration of x nM. The samples was mixed carefully by stirring. The final composition per one reaction was as follows:

Component	Final concentration	Volume
Buffer B	–	7 μL
dNTPs	x nM	0.75 μL
GamS	x nM	0.3 μL
Cell extract	–	5 μL
PEG-8000	x nM	0.9 μL
DNA	x nM	1.05 μL

The samples were incubated at 29°C for 8h in a thermocycler followed by a 4°C storage.

Results:

The efficiency of TXTL was evaluated by downstream experiments.

PCR (Platinum™ SuperFi™ II) of 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, and 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from @171, @172, and @173 for constructs 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES with high fidelity and efficiency.

gBlock	Construct	Primer	length	extension time
@171	590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES	146_F_gBlock & 147_R_gBlock	1.7 kb	1 min
@172	600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES	146_F_gBlock & 147_R_gBlock	1.7 kb	1 min
@173	610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES	146_F_gBlock & 147_R_gBlock	1.67	1 min

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA	10 ng	1 μL
DMSO	2.5 % (v/v)	0.5 µL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	5–10 sec	35
Annealing	58°C	20 sec
Extension	72°C	30 seconds/kb
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

E. coli re-transformation of 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, and 620_pBudCE4.1_MxEncA_STII-M

Goal:

Transformation was performed to introduce plasmid DNA construct250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, and 620_pBudCE4.1_MxEncA_STII-M into DH10β E. coli cells, allowing for the replication and expression of the desired genetic material.

Samples:

• 250.1 -> 250.4 Mini
• 250. 2 -> 250.5 Mini
• 260.2 .> 260.3 Mini
• 270.3 -> 270.5 Mini
• Lpn040 -> 620

Protocol:

For each construct, an aliquot of competent DH10β E. coli cells stored at −80 °C was thawed on ice. 3 µL of the desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 μL of room temperature LB, the mixture was then recovered for 60 min at 37 °C with shaking (#Heatblock). The mixture was centrifuged at 16000 x g for 30 seconds to remove the supernatant. The pellet was resuspended in 50 μl of LB and spread onto a selection plate and incubated overnight at 37°C.

Results:

PCR Clean-up (NEB) of @171, @172, and @173

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
@171	89.44	1.93	3.60
@172	113.67	1.92	3.09
@173	112.60	1.93	3.10

It was determined by gel electrophoresis that this experiment has to be redone:

from left to right: Ladder, 171, 172, 173, Ladder

Amplification of DNA using the phi29-XT RCA Kit for purified circular DNA: ligation product 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M) and 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)

Goal:

DNA of GA 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M) and 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M) (directly) was amplified using the phii29-XT RCA kit (NEB #E1603) for downstream applications.

Protocol:

Reactions with an end volume of 20 µL were prepared without enzyme as described in the table below. For each reaction, 4 µL of phi29-XT Reaction Buffer (5X) was added to achieve a final concentration of 1X, as well as 2 µL of Deoxynucleotide (dNTP) Solution Mix (10 mM) for a final concentration of 1 mM.  Furthermore, 2 µL of Exonuclease-Resistant Random Primers (500 µM) were added to achieve a final concentration of 50 µM, as well as X µL of the  DNA template for a final concentration of X. Nuclease-free water was used to bring the reaction to 18 µL.

Components	20 µL reaction	Final concentration
phi29-XT Reaction Buffer, 5X	4 µL	1X
Deoxynucleotide (dNTP) Solution Mix, 10 mM	2 µL	1 mM
Exonuclease-Resistant Random Primers, 500 µM	2 µL	50 µM
Circular DNA Template	up to 10 µL	variable
Nuclease-free Water	to 18 µL	N/A

Each reaction was mixed thoroughly by gentle pipetting or vortexing, followed by brief centrifuging.

After that, the reactions were incubated in a thermocycler (model) at 95 °C for 3 minutes, with a lid temperature of 100 °C, followed by cooling the reactions at room temperature.

The reactions were put on ice, and 2 µL of phi29-XT DNA Polymerase was added to each reaction, followed by mixing by gentle pipetting or vortexing, and brief centrifuging.

Lastly, the reactions were incubated in a thermocycler (model) at 42 °C for 2 hours, with a lid temperature of ≥ 75°C, followed by 10 minutes at 65 °C to inactivate the DNA polymerase.

The RCA products can be kept at 4 °C overnight or at  -20 °C for long-term storage.

Results:

The efficiency was evaluated by downstream experiments.

Amplification of DNA using the phi29-XT RCA Kit for purified circular DNA: direct GA 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)

Goal:

DNA of GA 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M) and 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M) (directly) was amplified using the phii29-XT RCA kit (NEB #E1603) for downstream applications.

Protocol:

Reactions with an end volume of 20 µL were prepared without enzyme as described in the table below. For each reaction, 4 µL of phi29-XT Reaction Buffer (5X) was added to achieve a final concentration of 1X, as well as 2 µL of Deoxynucleotide (dNTP) Solution Mix (10 mM) for a final concentration of 1 mM.  Furthermore, 2 µL of Exonuclease-Resistant Random Primers (500 µM) were added to achieve a final concentration of 50 µM, as well as X µL of the  DNA template for a final concentration of X. Nuclease-free water was used to bring the reaction to 18 µL.

Components	20 µL reaction	Final concentration
phi29-XT Reaction Buffer, 5X	4 µL	1X
Deoxynucleotide (dNTP) Solution Mix, 10 mM	2 µL	1 mM
Exonuclease-Resistant Random Primers, 500 µM	2 µL	50 µM
Circular DNA Template	up to 10 µL	variable
Nuclease-free Water	to 18 µL	N/A

Each reaction was mixed thoroughly by gentle pipetting or vortexing, followed by brief centrifuging.

After that, the reactions were incubated in a thermocycler (model) at 95 °C for 3 minutes, with a lid temperature of 100 °C, followed by cooling the reactions at room temperature.

The reactions were put on ice, and 2 µL of phi29-XT DNA Polymerase was added to each reaction, followed by mixing by gentle pipetting or vortexing, and brief centrifuging.

Lastly, the reactions were incubated in a thermocycler (model) at 42 °C for 2 hours, with a lid temperature of ≥ 75°C, followed by 10 minutes at 65 °C to inactivate the DNA polymerase.

The RCA products can be kept at 4 °C overnight or at  -20 °C for long-term storage.

Results:

The efficiency was evaluated by downstream experiments.

PCR Clean-up (NEB) of linearization product of 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M) and 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)	201	1.96	2.77
500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)	195	1.94	2.91

E. coli re-transformation with 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP .6

Goal:

Transformation was performed to introduce plasmid DNA construct 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP .6 into DH10β E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

For each construct, an aliquot of competent DH10β E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells (1–5 μL) and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 μL of room temperature SOC, the mixture was then recovered for 30–60 min at 37 °C with shaking (#Heatblock). The mixture was centrifuged at 16000 x g for 30 seconds to remove the supernatant. The pellet was resuspended in 50 μl of LB and spread onto a selection plate and incubated overnight at 37°C.

Results:

Amplification of DNA using the phi29-XT RCA Kit for purified circular DNA: direct GA 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M) and 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)

Goal:

DNA of GA 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M) and 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M) (directly) was amplified using the phii29-XT RCA kit (NEB #E1603) for downstream applications.

Protocol:

Reactions with an end volume of 20 µL were prepared without enzyme as described in the table below. For each reaction, 4 µL of phi29-XT Reaction Buffer (5X) was added to achieve a final concentration of 1X, as well as 2 µL of Deoxynucleotide (dNTP) Solution Mix (10 mM) for a final concentration of 1 mM.  Furthermore, 2 µL of Exonuclease-Resistant Random Primers (500 µM) were added to achieve a final concentration of 50 µM, as well as X µL of the  DNA template for a final concentration of X. Nuclease-free water was used to bring the reaction to 18 µL.

Components	20 µL reaction	Final concentration
phi29-XT Reaction Buffer, 5X	4 µL	1X
Deoxynucleotide (dNTP) Solution Mix, 10 mM	2 µL	1 mM
Exonuclease-Resistant Random Primers, 500 µM	2 µL	50 µM
Circular DNA Template	up to 10 µL	variable
Nuclease-free Water	to 18 µL	N/A

Each reaction was mixed thoroughly by gentle pipetting or vortexing, followed by brief centrifuging.

After that, the reactions were incubated in a thermocycler (model) at 95 °C for 3 minutes, with a lid temperature of 100 °C, followed by cooling the reactions at room temperature.

The reactions were put on ice, and 2 µL of phi29-XT DNA Polymerase was added to each reaction, followed by mixing by gentle pipetting or vortexing, and brief centrifuging.

Lastly, the reactions were incubated in a thermocycler (model) at 42 °C for 2 hours, with a lid temperature of ≥ 75°C, followed by 10 minutes at 65 °C to inactivate the DNA polymerase.

The RCA products can be kept at 4 °C overnight or at  -20 °C for long-term storage.

Results:

The efficiency was evaluated by downstream experiments.

PCR Clean-up (NEB) of GA for 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M) and 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Amplification of DNA using the phi29-XT RCA Kit for purified circular DNA for 330_LET_CLS-XaCS-BmTyr, 340_LET_CLS-XaCS-BmTyr(E195S+A221V), 350_LET_CLS-XaCS-BmTyr(R209S+M215E+V218M), 360_LET_CLS-XaCS-BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C), 370_LET_CLS-XaCS-cpBmTyr(201), 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201), 440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466), 460_LET_CLS-XaCS-LsTyr, 470_LET_CLS-XaCS-SkMelC1(33-124),

480_LET_CLS-XaCS-SkMelC2, 490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518), 510_LET_CLS-XaCS-SavMelC1(27-118), 520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F), 530_LET_CLS-XaCS-SavMelC2 and 540_LET_CLS-XaCS-SavMelC2(I42Y)

Goal:

DNA of 330_LET_CLS-XaCS-BmTyr, 340_LET_CLS-XaCS-BmTyr(E195S+A221V), 350_LET_CLS-XaCS-BmTyr(R209S+M215E+V218M), 360_LET_CLS-XaCS-BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C), 370_LET_CLS-XaCS-cpBmTyr(201), 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201), 440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466), 460_LET_CLS-XaCS-LsTyr, 470_LET_CLS-XaCS-SkMelC1(33-124), 480_LET_CLS-XaCS-SkMelC2, 490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518), 510_LET_CLS-XaCS-SavMelC1(27-118), 520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F), 530_LET_CLS-XaCS-SavMelC2 and 540_LET_CLS-XaCS-SavMelC2(I42Y) was amplified using the phii29-XT RCA kit (NEB #E1603) for downstream applications.

Protocol:

Reactions with an end volume of 20 µL were prepared without enzyme as described in the table below. For each reaction, 4 µL of phi29-XT Reaction Buffer (5X) was added to achieve a final concentration of 1X, as well as 2 µL of Deoxynucleotide (dNTP) Solution Mix (10 mM) for a final concentration of 1 mM.  Furthermore, 2 µL of Exonuclease-Resistant Random Primers (500 µM) were added to achieve a final concentration of 50 µM, as well as X µL of the  DNA template for a final concentration of X. Nuclease-free water was used to bring the reaction to 18 µL.

Components	18 µL reaction	Final concentration
phi29-XT Reaction Buffer, 5X	4 µL	1X
Deoxynucleotide (dNTP) Solution Mix, 10 mM	2 µL	1 mM
Exonuclease-Resistant Random Primers, 500 µM	2 µL	50 µM
Circular DNA Template	2 µL	variable
Nuclease-free Water	to 18 µL	N/A

Each reaction was mixed thoroughly by gentle pipetting or vortexing, followed by brief centrifuging.

After that, the reactions were incubated in a thermocycler (model) at 95 °C for 3 minutes, with a lid temperature of 100 °C, followed by cooling the reactions at room temperature.

The reactions were put on ice, and 2 µL of phi29-XT DNA Polymerase was added to each reaction, followed by mixing by gentle pipetting or vortexing, and brief centrifuging.

Lastly, the reactions were incubated in a thermocycler (model) at 42 °C for 4 hours, with a lid temperature of ≥ 75°C, followed by 10 minutes at 65 °C to inactivate the DNA polymerase.

The RCA products can be kept at 4 °C overnight or at  -20 °C for long-term storage.

Results:

The efficiency was evaluated by downstream experiments.

Sanger sequencing of Maxis of 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP

Goal:

To confirm the nucleotide sequences of

230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP

Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a (partial) match between the experimental and expected sequences. In addition, the sequencing data showed (predominantly) a high quality score and a continuous read length of over 1 kb.

Sample	Primer	Validation
230	CMV	Success
230	WPRE	Success
250	CMV	Point mutations in the insert
250	WPRE	Point mutations in the insert
260	CMV	Point mutations and deletions in the insert
260	WPRE	Point mutations and deletions in the insert
270	CMV	Success
270	WPRE	No priming

GA clean up: 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201)

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from GA reactions 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201) for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
380_LET_CLS-XaCS-NBmTyr(85)-P4	6,44	2,58	-0,31
390_LET_CLS-XaCS-P3-CBmTyr(85)	8,04	1,83	-0,50
400_LET_CLS-XaCS-NBmTyr(157)-P4	8,12	1,69	-0,37
410_LET_CLS-XaCS-P3-CBmTyr(157)	10,04	1,88	-0,56
420_LET_CLS-XaCS-NBmTyr(201)-P4	11,24	1,45	-0,23
430_LET_CLS-XaCS-P3-CBmTyr(201)	7,04	2,00	-0,57

PCR Clean-up (NEB): @156, @157, @162 and @166 for constructs 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201), 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M), 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M), LET-P3, and P4-LET

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from GA reactions @156, @157, @162 and @166 for constructs 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201), 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M), 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M), LET-P3, and P4-LET

, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

	Sample	Concentration [ng/μL]	A260/280	A260/230
1	450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)	141	1.95	2.57
2	500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)	106	1.93	2.85
3	390_LET_CLS-XaCS-P3-CBmTyr(85), 410_LET_CLS-XaCS-P3-CBmTyr(157), 430_LET_CLS-XaCS-P3-CBmTyr(201)	52	1.95	19.81
4	380_LET_CLS-XaCS-NBmTyr(85)-P4, 400_LET_CLS-XaCS-NBmTyr(157)-P4, 420_LET_CLS-XaCS-NBmTyr(201)-P4	151	1.95	2.96
5	380_LET_CLS-XaCS-NBmTyr(85)-P4	117	1.95	3.44
6	390_LET_CLS-XaCS-P3-CBmTyr(85)	193	1.94	1.91
7	400_LET_CLS-XaCS-NBmTyr(157)-P4	192	1.98	1.93
8	410_LET_CLS-XaCS-P3-CBmTyr(157)	not performed	not perfomed	not performed due to evaporation during pcr
9	420_LET_CLS-XaCS-NBmTyr(201)-P4	187	1.94	2.97
10	430_LET_CLS-XaCS-P3-CBmTyr(201)	148	1.94	3.06

PCR Clean-up (NEB) for 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201), 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M), and 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from GA reactions (380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201), 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M), and 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)), for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Agarose Gel Electrophoresis & Gel-Ex for @156, @157, @162 and @166 for constructs 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201), 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M), 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M), LET-P3, and P4-LET

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Protocol:

Agarose gel electrophoresis was performed using a 2% w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model) and cut out from the gel. Gel extraction following the ... protocol was performed on the cut out gel fragments, to isolate the DNA.

Results:

The following samples were loaded into the gel from left to right:

from PCR: 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M); 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M); 390_LET_CLS-XaCS-P3-CBmTyr(85), 410_LET_CLS-XaCS-P3-CBmTyr(157), 430_LET_CLS-XaCS-P3-CBmTyr(201); 380_LET_CLS-XaCS-NBmTyr(85)-P4, 400_LET_CLS-XaCS-NBmTyr(157)-P4, 420_LET_CLS-XaCS-NBmTyr(201)-P4; 380_LET_CLS-XaCS-NBmTyr(85)-P4; 390_LET_CLS-XaCS-P3-CBmTyr(85);400_LET_CLS-XaCS-NBmTyr(157)-P4;410_LET_CLS-XaCS-P3-CBmTyr(157);420_LET_CLS-XaCS-NBmTyr(201)-P4;430_LET_CLS-XaCS-P3-CBmTyr(201)

results from gel-extraction:

Sample	ng/µl	260/280	260/230
450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)	35.4	2.86	0.1
500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)	22.2	1.92	0.06
390_LET_CLS-XaCS-P3-CBmTyr(85),410,430_LET_CLS-XaCS-P3-CBmTyr(201)	24.0	1.92	0.2
390_LET_CLS-XaCS-P3-CBmTyr(85)	27.3	1.83	0.16
400_LET_CLS-XaCS-NBmTyr(157)-P4	33.8	1.79	0.43

PCR Clean-up (NEB): 330_LET_CLS-XaCS-BmTyr, 340_LET_CLS-XaCS-BmTyr(E195S+A221V), 350_LET_CLS-XaCS-BmTyr(R209S+M215E+V218M), 360_LET_CLS-XaCS-BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C), 370_LET_CLS-XaCS-cpBmTyr(201), 440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466), 460_LET_CLS-XaCS-LsTyr, 470_LET_CLS-XaCS-SkMelC1(33-124), 480_LET_CLS-XaCS-SkMelC2, 490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518), 510_LET_CLS-XaCS-SavMelC1(27-118), 520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F), 530_LET_CLS-XaCS-SavMelC2, and 540_LET_CLS-XaCS-SavMelC2(I42Y)

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from GA reactions (330_LET_CLS-XaCS-BmTyr, 340_LET_CLS-XaCS-BmTyr(E195S+A221V), 350_LET_CLS-XaCS-BmTyr(R209S+M215E+V218M), 360_LET_CLS-XaCS-BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C), 370_LET_CLS-XaCS-cpBmTyr(201), 440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466), 460_LET_CLS-XaCS-LsTyr, 470_LET_CLS-XaCS-SkMelC1(33-124), 480_LET_CLS-XaCS-SkMelC2, 490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518), 510_LET_CLS-XaCS-SavMelC1(27-118), 520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F), 530_LET_CLS-XaCS-SavMelC2, and 540_LET_CLS-XaCS-SavMelC2(I42Y)), for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Gibson Assembly 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201), 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M), and 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)

Goal:

Gibson assembly was performed to assemble the @156, LET-P3, P4-LET vector to produce 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201), 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M), and 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M).

Protocol:

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Construct	Vector	Insert
380_LET_CLS-XaCS-NBmTyr(85)-P4	P4-LET	PCR5
390_LET_CLS-XaCS-P3-CBmTyr(85)	LET-P3	PCR6
400_LET_CLS-XaCS-NBmTyr(157)-P4	P4-LET	PCR7
410_LET_CLS-XaCS-P3-CBmTyr(157)	LET-P3	PCR8
420_LET_CLS-XaCS-NBmTyr(201)-P4	P4-LET	PCR9
430_LET_CLS-XaCS-P3-CBmTyr(201)	LET-P3	PCR10
450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)	@156	PCR1
500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)	@156	PCR2

Components	Volume
NEBuilder HiFi DNA Assembly Master Mix	10 µL
Vector	20 ng = 2µL
Insert	x ng
Nuclease-free Water	x μL
Total	20 µL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of Gibson assembly was evaluated by downstream experiments.

QIAGEN Maxiprep 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP .a1, 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP .2, 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K) .a1, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP .3

Things to do before starting:

• Before use, centrifuge RNase A briefly, and then add into Buffer P1 to obtain a final concentration of 100 μg/mL
• Check Buffer P2 for SDS precipitation due to low storage temperatures. If necessary, dissolve the SDS by warming to 37°C.
• Prechill Buffer P3 on ice.
• If not prepared: Add the provided LyseBlue reagent to Buffer P1 and mix before use. Use 1 vial LyseBlue reagent per bottle Buffer P1 for a final dilution of 1:1000 (e.g., 10 µL LyseBlue into 10 mL Buffer P1).

Maxi prep:

1. Harvest the bacterial cells of the culture 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP .a1, 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP .1, 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K) .a1, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP .3 by centrifugation at 6000 x g for 15 min at 4°C. (If you wish to stop the protocol and continue later, freeze the cell pellets at −30 to −15°C.
2. Resuspend the bacterial pellet in 10 mL of Buffer P1. Ensure that RNase A has been added to Buffer P1, shake the buffer bottle before use. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
3. Add 10 mL of Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4−6 times, and incubate at room temperature for 5 min (not longer!). Do not vortex because this will result in shearing of genomic DNA! After use, the bottle containing Buffer P2 should be closed immediately to avoid acidification from CO2 in the air.
4. Add 10 ml prechilled Buffer P3, mix thoroughly by vigorously inverting 4–6 times. Incubate on ice for 20 min. If using LyseBlue reagent, mix the solution until it is colorless.
5. Centrifuge at ≥20,000 x g for 135 min at 4°C.
6. Equilibrate a QIAGEN-tip 500 by applying 10 ml Buffer QBT, and allow column to empty by gravity flow.
7. Apply the supernatant from step 5 to the QIAGEN-tip and allow it to enter the resin by gravity flow.
8. Wash the QIAGEN-tip with 2 x 30 ml Buffer QC. Allow Buffer QC to move through the QIAGEN-tip by gravity flow.
9. Elute DNA with 15 ml Buffer QF into a clean 50 ml vessel. For constructs larger than 45 kb, prewarming the elution buffer to 65°C may help to increase the yield.
10. Precipitate DNA by adding 10.5 ml (0.7 volumes) room temperature isopropanol to the eluted DNA and mix. Centrifuge at ≥15,000 x g for 2 h at 4°C. Carefully decant the supernatant. If possible, try to transfer the pellet into a 2 mL Eppendorf
11. Wash the DNA pellet with 5 ml room-temperature 70% ethanol and centrifuge at ≥15,000 x g for 10 min. Carefully decant supernatant. If you have your pellet in a 2 mL Eppendorf, wash twice with 2 mL 70% ethanol.
12. Air-dry pellet for 5–10 min and redissolve DNA in a suitable volume of appropriate buffer (e.g., TE buffer, pH 8.0, or 10 mM Tris·Cl, pH 8.5). Start with 200 µL buffer, measure the concentration with the Nanodrop, and add more buffer volume if necessary. Write down the plasmid concentration and the ratios 260/280 and 260/230.
13. Make a 200 µL working stock solution with a plasmid concentration of 100 ng/µL
14. Store the working stock solution in the fridge and the original stock solution in the freezer

Results:

Sample	Concentration ng/μl	260/280	260/230
260.3	2849	1.93	2.15
270.1	227	1.93	2.18
230.a1	31	1.88	7.32
250.2	n.a.	n.a.	n.a.

Sanger re-sequencing of 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

To confirm the nucleotide sequences of 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4, Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a (partial) match between the experimental and expected sequences. In addition, the sequencing data showed (predominantly) a high quality score and a continuous read length of over 1 kb.

Sample	Primer	Validation
220 Maxi	WPRE	confirmed
240 Maxi	WPRE	confirmed
250.2 Mini	WPRE	confirmed
250.2 Mini	mTagBFP	no priming
260.2 Mini	WPRE	confirmed
260.2 Mini	mTagBFP	confirmed
260.2 Mini	CMV	mutation
260.6	WPRE	confirmed
260.6	mTagBFP	silent mutation
260.6	CMV	confirmed

Agarose Gel Electrophoresis of analyical digest of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, and 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Protocol:

Agarose gel electrophoresis was performed using a 1 % w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. 4 µL of DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), SDS; New England Biolabs) were loaded into the wells of the gel. 8 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right: L-empty-270.2-270.3-270.4-280.3-280.4-280.5-280.6-290.3-290.4-290.5-290.6-L Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

Analytical digest of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) with MIuI-HF and KpnI-HF

Goal:

Restriction digest of the 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP .a1, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP.a2, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP .A3, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP .A4, 280_pcDNAT7v1_RS20-cpEFGP-CaM .3, 280_pcDNAT7v1_RS20-cpEFGP-CaM .4, 280_pcDNAT7v1_RS20-cpEFGP-CaM .5, and 280_pcDNAT7v1_RS20-cpEFGP-CaM .6 minipreps with KpnI HF and Mlul HF enzymes, as well as 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) .3, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) .4, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) .5, and 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) .6 minipreps with Mlul HF enzyme for checking purposes.

Protocol:

Restriction digest was carried out using the KpnI enzyme (R3142,  New England Biolabs) and MluI enzyme (R3198, New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 1 μL of rCutSmart buffer (B7204, New England Biolabs) and 1 U of the respective enzymes in a PCR tube with a total reaction volume of 10 μL.

Master mix for the digestion of 270 and 280 was prepared as follows:

Component	Volume
Restriction enzyme MIuI	2 μL
Restriction enzyme KpnI	2 µL
rCutSmart buffer	10 μL
Nuclease-free Water	36 μL

Master mix for the digestion of 290 was prepared as follows:

Component	Volume
Restriction enzyme MIuI	1 μL
rCutSmart buffer	5 μL
Nuclease-free Water	34 μL

5 μl of the master mix was supplemented with 5 μl of template DNA for the digest of 280. For the digest of 270, 2 µL of template DNA was diluted with 3 µL nuclease-free water before adding 5 µL of master mix. 8 µL of the master mix was supplemented with 2 µL of template DNA for the digest of 290. The samples were mixed by pipetting and incubated at 37 °C for 20 minutes.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

E. coli transformation 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP .a1 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K) .a1 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP .2

Goal:

Transformation was performed to introduce plasmid DNA constructs 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP .a1, 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP .2, 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K) .a1 into DH10β E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

For each construct, an aliquot of competent DH10β E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells (1–5 μL) and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 μL of room temperature SOC, the mixture was then recovered for 30–60 min at 37 °C with shaking (#Heatblock). The mixture was centrifuged at 16000 x g for 30 seconds to remove the supernatant. The pellet was resuspended in 100 μl of LB and 50 uL were spread onto a selection plate and 50 uL were added to 200 ml LB medium for later maxi prep and incubated overnight at 37°C.

Results:

Sanger sequencing 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

To confirm the nucleotide sequences of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4, Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a partial match between the experimental and expected sequences. In addition, the sequencing data showed predominantly a high quality score and a continuous read length of over 1 kb.

Sample	Primer	Validation
280.1	CMV-Forward	Success
280.1	T7-Term	Success
280.2	CMV-Forward	Wrong primer
280.2	T7-Term	Wrong primer
290.1	CMV-Forward	Wrong primer
290.1	T7-Term	Wrong primer
290.2	CMV-Forward	Wrong primer
290.2	T7-Term	Wrong primer
310.1	CMV-Forward	Wrong primer
310.1	T7-Term	Wrong primer
310.2	CMV-Forward	Wrong primer
310.2	T7-Term	Wrong primer
270.1	CMV-Forward	Wrong primer
270.1	T7-Term	Wrong primer
270.2	CMV-Forward	Wrong primer
270.2	T7-Term	Wrong primer

Agarose Gel Electrophoresis 320_pcDNAT7v2, 200_pcDNAT7v1

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Protocol:

Agarose gel electrophoresis was performed using a 1.0% w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5–10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right: A,B,C,D,E,F, 200_pcDNAT7v1, dA, dB, dC, dD, dE, dF Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

MluI analytical digest RCA 200_pcDNAT7v1 320_pcDNAT7v2 under different conditions

Goal:

Restriction digest of the 320_pcDNAT7v2 DNA 2h, 320_pcDNAT7v2 DNA 4h, 200_pcDNAT7v1 DNA 4h, 320_pcDNAT7v2 colony 2h, 320_pcDNAT7v2 colony 4h, neg control 4h with MluI enzyme, for troubleshooting RCA.

Construct name + condition	320 DNA 2h	320 DNA 4h	200 DNA 4h	320 colony 2h	320 colony 4h	neg control 4h with MluI enzyme
Sample name	A	B	C	D	E	F

Protocol:

Restriction digest was carried out using the MluI enzyme(s) (R3198,  New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 1 hour.

Component	Volume
Restriction enzyme	10 U (1 μL)
rCutSmart buffer	5 μL
DNA	1 μg
Nuclease-free Water	to 50 μL

Optionally, the reaction was stopped using heat inactivation (80 °C, 20 minutes). The products were then stored at –20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

Agarose Gel Electrophoresis of Analytical MluI digestion of 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 320_pcDNAT7v2, 550_pCas9_sgAAVS1_Cas9-HA-2xNLS-i53, 560_pAAVS1_CAGp-IL7Ra-IRES-BSD, 560_pAAVS1_CAGp-IL7Ra-IRES-BSD-2

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Protocol:

Agarose gel electrophoresis was performed using a 1 % w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5–10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right: 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 320_pcDNAT7v2, 560_pAAVS1_CAGp-IL7Ra-IRES-BSD .1, 550_pCas9_sgAAVS1_Cas9-HA-2xNLS-i53, 560_pAAVS1_CAGp-IL7Ra-IRES-BSD .2 Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

Analytical Restriction digest with MluI of 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 320_pcDNAT7v2, 550_pCas9_sgAAVS1_Cas9-HA-2xNLS-i53, 560_pAAVS1_CAGp-IL7Ra-IRES-BSD, 560_pAAVS1_CAGp-IL7Ra-IRES-BSD-2

Goal:

Restriction digest of the

210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 320_pcDNAT7v2, 550_pCas9_sgAAVS1_Cas9-HA-2xNLS-i53, 560_pAAVS1_CAGp-IL7Ra-IRES-BSD .1, 560_pAAVS1_CAGp-IL7Ra-IRES-BSD .2

with MluI enzyme(s), for agarose gel electrophoresis

Protocol:

Restriction digest was carried out using the MluI enzyme(s) (R3198,  New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 1 hour.

Component	Volume
Restriction enzyme	10 U (1 μL)
rCutSmart buffer	5 μL
DNA	1 μg
Nuclease-free Water	to 50 μL

Optionally, the reaction was stopped using heat inactivation (80 °C, 20 minutes). The products were then stored at –20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

Sanger sequencing of 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 320_pcDNAT7v2, 550_pCas9_sgAAVS1_Cas9-HA-2xNLS-i53, 560_pAAVS1_CAGp-IL7Ra-IRES-BSD, 560_pAAVS1_CAGp-IL7Ra-IRES-BSD-2

Goal:

To confirm the nucleotide sequences of 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 320_pcDNAT7v2, 550_pCas9_sgAAVS1_Cas9-HA-2xNLS-i53, 560_pAAVS1_CAGp-IL7Ra-IRES-BSD .1 and 560_pAAVS1_CAGp-IL7Ra-IRES-BSD .2 Sanger sequencing was performed by using Genewiz, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 50 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis ... a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Sample	Barcode	Primer	Validation
210	06817519	T7	confirmed
210	06817520	T7-Term	confirmed
220	06817521	T7	no priming
220	06817522	T7-Term	confirmed
230	06817523	T7	confirmed
230	06817524	T7-Term	confirmed
240	06817525	T7	no priming
240	06817526	T7-Term	confirmed
250	06817527	T7	no priming
250	06817528	T7-Term	no insert
290	06817529	T7	no priming
290	06817530	T7-Term	no insert
320	06817531	T7	wrong sequencing method
320	06817532	T7-Term	wrong sequencing method
550.1	06817533	T7	wrong sequencing method
550.1	06817534	T7-Term	wrong sequencing method
560.1	06817535	T7	wrong sequencing method
560.1	06817536	T7-Term	wrong sequencing method
560.2	06817537	T7	wrong sequencing method
560.2	06817538	T7-Term	wrong sequencing method

KLD of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP v2 & v3

Goal:

KLD reaction was performed to assemble construct 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP v2 and v3 from PCR product 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP.

Protocol:

KLD reaction was performed using the KLD Enzyme Mix (M0554, New England Biolabs), consisting of a kinase, a ligase, and a DpnI restriction enzyme, according to the manufacturer's instructions. For each reaction, KLD reaction buffer, KLD enzyme mix, nuclease-free water, and the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 μL
KLD Reaction Buffer (2x)	5 μL
KLD Enzyme Mix (10x)	1 μL
Nuclease-free water	3 μL
Total	10 μL

The sample was incubated at room temperature (25 °C) for 5–10 minutes. Optionally, to increase the efficiency of the DpnI digestion, the mixture was additionally incubated at 37 °C for 30–60 minutes. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of KLD reaction was evaluated by downstream experiments.

DpnI digest of 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 v2 & v3

Goal:

Restriction digest of the backbone PCR fragments of 200_pcDNAT7v1, @153, @154, @155 with DpnI enzyme, for downstream cloning of 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 v2 and v3 for each sample.

Protocol:

Restriction digest was carried out using the DpnI enzyme (R0176, New England Biolabs)  according to the manufacturer's instructions. Template DNA was supplied in an amount of 1.5 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of DpnI in a 1.5 mL microcentrifuge tube. The sample was mixed by pipetting and incubated at 37°C for 30 min.

Component	Volume
Restriction enzyme	10 U (1 μL)
rCutSmart buffer	2.5 μL
DNA	1.5 μg (8 μL)
Nuclease-free Water	to 25 μL

The reaction was then stopped using heat inactivation (80°C, 10 minutes). The product was stored at -20°C or immediately used for downstream experiments.

Based on the measured concentration, only half of the amount of Enzyme, Water and Buffer were used for 153 (280).

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

PCR Clean-up (NEB) of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, @ 290, 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 v2 & v3

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, @ 290, 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 v2 and v3 for each sample for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
280 v2	63	1.6	3.7
280 v3	219	1.9	2.7
290 v2	109	2.0	3.7
290 v3	232	2.0	2.6
310 v2	51	1.9	11.3
310 v3	88	2.0	4.0
270 v2	41	1.5	11.0
270 v3	56	1.9	6.9

Agarose Gel Electrophoresis 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP 280_pcDNAT7v1_RS20-cpEFGP-CaM 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 v2 & v3

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Protocol:

Agarose gel electrophoresis was performed using a 1% w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5–10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right: Marker, 280_pcDNAT7v1_RS20-cpEFGP-CaM (v2 & v3), 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) (v2 & v3), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 (v2 & v3), 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP (v2 & v3) Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

Amplification of DNA using the phi29-XT RCA Kit of 320_pcDNAT7v2 colonies

Goal:

DNA of mid- or high-copy number plasmids 320_pcDNAT7v2 was amplified using the phi29-XT RCA kit (NEB #E1603) for downstream applications.

Protocol:

For each sample, a sterile pipette tip was used to pick a bacterial colony from an agar plate containing 200_pcDNAT7v1. The colony was resuspended in 25 µL nuclease-free water in a PCR tube.

The cells were lysed by incubation in a thermocycler (model) at 95 °C for 3 minutes, with a lid temperature of 100 °C. Afterwards, the samples were transferred to ice.

Reactions with an end volume of 20 µL were prepared as described in the table below. For each reaction, 4 µL of phi29-XT Reaction Buffer (5X) was added to achieve a final concentration of 1X, as well as 2 µL of Deoxynucleotide (dNTP) Solution Mix (10 mM) for a final concentration of 1 mM.  Furthermore, 2 µL of Exonuclease-Resistant Random Primers (500 µM) were added to achieve a final concentration of 50 µM, as well as 2 µL of phi29-XT DNA Polymerase (10X) for a final concentration of 1X. 10 µL of the lysed cells was added. Nuclease-free water was used to bring the reaction to 18 µL.

Components	20 µL reaction	Final concentration	pD	pE	pF
phi29-XT Reaction Buffer, 5X	4 µL	1X	4 µL	4 µL	4 µL
Deoxynucleotide (dNTP) Solution Mix, 10 mM	2 µL	1 mM	2 µL	2 µL	2 µL
Exonuclease-Resistant Random Primers, 500 µM	2 µL	50 µM	2 µL	2 µL	2 µL
phi29-XT DNA Polymerase, 10X	2 µL	1X	2 µL	2 µL	2 µL
Lysed cells (320_pcDNAT7v2)	10 µL	N/A	10 µL	10 µL	–
Nuclease-free Water	– µL	N/A	–	–	10 µL
42 °C incubation time			2 h	4 h	4 h

Each reaction was mixed thoroughly by gentle pipetting or vortexing, followed by brief centrifuging.

Lastly, the reactions were incubated in a thermocycler (model) at 42 °C for pD) 2 hours pE) 4 hours, with a lid temperature of ≥ 75°C, followed by 10 minutes at 65 °C to inactivate the DNA polymerase.

The RCA products can be kept at 4 °C overnight or at  -20 °C for long-term storage.

Results:

PCR (Platinum™ SuperFi™ II) 280_pcDNAT7v1_RS20-cpEFGP-CaM 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP v2 & v3

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from @153, @154, and @155, 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP for constructs 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP with high fidelity and efficiency according to two different protocols.

Protocol 1

Constructs:

Sample name	Template	Primers	kb	Elongation time
280v2	153	282_R_CaM-NcoI_pcDNA & 281_F_SacI-RS20_T7p	1.3	45 sec
290v2	154	281_F_SacI-RS20_T7p & 292_R_CTEVp-Stop-NcoI_T7tt	1.5	55 sec
310v2	155	281_F_SacI-RS20_T7p & 312_R_P4-Stop-NcoI_T7tt	0.7	30 sec
270v2	250	271_F_tTA-(G4S)2L & 272_R_TetR	8.5	4 min 25 sec

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA	10 ng	x μL
Nuclease-free water	–	4 μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	5–10 sec	35
Annealing	60°C	10 sec
Extension	72°C	15–30 seconds/kb
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

Protocol 2

Constructs:

Construct	Template	Primers	kb	Elongation time
280v3	153	282_R_CaM-NcoI_pcDNA & 281_F_SacI-RS20_T7p	1.3	45 sec
290v3	154	281_F_SacI-RS20_T7p & 292_R_CTEVp-Stop-NcoI_T7tt	1.5	55 sec
310v3	155	281_F_SacI-RS20_T7p & 312_R_P4-Stop-NcoI_T7tt	0.7	30 sec
270v3	250	271_F_tTA-(G4S)2L & 272_R_TetR	8.5	4 min 25 sec

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	25-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	12.5 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2.5 μL
Template DNA	–	2 μL
Nuclease-free water	–	8 μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	5–10 sec	35
Annealing	60°C	10 sec
Extension	72°C	15–30 seconds/kb
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

DpnI digest of 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

Restriction digest of the backbone PCR fragments of 200_pcDNAT7v1, @153, @154, @155 with DpnI enzyme, for downstream cloning of 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4.

Protocol:

Restriction digest was carried out using the DpnI enzyme (R0176, New England Biolabs)  according to the manufacturer's instructions. Template DNA was supplied in an amount of 1.5 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of DpnI in a 1.5 mL microcentrifuge tube. The sample was mixed by pipetting and incubated at 37°C for 30 min.

Component	Volume
Restriction enzyme	10 U (1 μL)
rCutSmart buffer	2.5 μL
DNA	1.5 μg (8 μL)
Nuclease-free Water	to 25 μL

The reaction was then stopped using heat inactivation (80°C, 10 minutes). The product was stored at -20°C or immediately used for downstream experiments.

Based on the measured concentration, only half of the amount of Enzyme, Water and Buffer were used for @153.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

PCR Clean-up (NEB) of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, @ 290, 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range for constructs 200_pcDNAT7v1 (280_pcDNAT7v1_RS20-cpEFGP-CaM), 200_pcDNAT7v1 (290), @154 (290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)), 200_pcDNAT7v1 (310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4) and @155 (310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4).

Sample	Concentration [ng/μL]	A260/280	A260/230
200 (280)	107	1.9	3.8
153 (280)	41	1.9	-4.2
200 (290)	140	1.9	3.3
154 (290)	107	1.9	3.7
200 (310)	154	2.0	3.1
155 (310)	152	1.9	3.1
250 (270)	29	1.9	-5.6

PCR (Platinum™ SuperFi™ II) 280_pcDNAT7v1_RS20-cpEFGP-CaM 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 200_pcDNAT7v1, @153, @154, and @155 for constructs 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 with high fidelity and efficiency.

Constructs:

Construct	Template	Primers	kb	Elongation time
280	200	254_R_T7p-SacI_Kozak-CD4SP & 283_F_NcoI-pcDNA_CaM	6	3 min 15 sec
	153	282_R_CaM-NcoI_pcDNA & 281_F_SacI-RS20_T7p	1.3	55 sec
290	200	293_F_NcoI-T7tt_CTEVp-Stop & 254_R_T7p-SacI_Kozak-CD4SP	6	3 min 15 sec
	154	281_F_SacI-RS20_T7p & 292_R_CTEVp-Stop-NcoI_T7tt	1.5	55 sec
310	200	313_F_NcoI-T7tt_P4-Stop & 254_R_T7p-SacI_Kozak-CD4SP	6	3 min 15 sec
	155	281_F_SacI-RS20_T7p & 312_R_P4-Stop-NcoI_T7tt	0.7	30 sec
270	250	271_F_tTA-(G4S)2L & 272_R_TetR	8.5	4 min 25 sec

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA	10 ng	X μL
Nuclease-free water	–	4 μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	5–10 sec	35
Annealing	60°C	10 sec
Extension	72°C	15–30 seconds/kb
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

E. coli transformation with 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP

Goal:

Transformation was performed to introduce plasmid DNA constructs 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP sample 260.3 (1) into DH10β E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

For each construct, an aliquot of competent DH10β E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells (5 μL) and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 μL of room temperature LB Medium, the mixture was then recovered for 30–60 min at 37 °C with shaking (#Heatblock). The mixture was centrifuged at 16000 x g for 30 seconds to remove the supernatant. The pellet was resuspended in 50 μl of LB and spread onto a selection plate and incubated overnight at 37°C.

Results:

Analytical digest of 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 with BsmBI-v2

Goal:

Restriction digest of the 310.5, 310.6, 310.7, 310.8, 310.9, 310.10, 310.11 and 310.12 minipreps of the 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 with BsmBI-v2 HF enzyme, for checking purposes.

Protocol:

Restriction digest was carried out using the BsmBI-v2 enzyme (R0739,  New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 1 μL of NEBuffer r3.1 (B6003, New England Biolabs) and 1 U of the respective enzymes in a PCR tube with a total reaction volume of 10 μL.

Master mix for the digestion was prepared as follows:

Component	Volume
Restriction enzyme	2 μL
NEBuffer r3.1	10 μL
Nuclease-free Water	78 μL

9 μl of the master mix was supplemented with 1 μl of template DNA for each reaction. The samples were mixed by pipetting and incubated at 37 °C for 30 minutes.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

Agarose Gel Electrophoresis of PCR for 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K), and 320_pcDNAT7v2

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

No.	Amplicon size	Cloning intermediate
31	7042 bp	220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S)
32	7042 bp	230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)
33	7804 bp	240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP
34	8455 bp	270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP
35	7549 bp	300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K)
36	6051 bp	320_pcDNAT7v2

Protocol:

Agarose gel electrophoresis was performed using a 1.0% w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples (2 μL of sample with 3 μL of H₂O) mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 2 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 150 V was applied, and the DNA fragments were allowed to migrate through the gel for 20 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right: NEB 1 kB ladder, PCR31.1, PCR31.2, PCR32.1–PCR32.4, PCR33.1–PCR33.4, PCR34.1–PCR34.4; NEB 1 kB ladder, PCR30, PCR35.1–PCR35.3 Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

Gibson Assembly of 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

Gibson assembly was performed to assemble the backbone 200_pcDNAT7v1 (samples 20–24) and insert (samples 25–30) amplicons to produce 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 constructs.

Protocol:

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50–100 ng of vector with 2–5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Components	Volume
NEBuilder HiFi DNA Assembly Master Mix	10 μL
Vector	1 μL
Insert	3 μL
Nuclease-free Water	6 μL
Total	20 μL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of Gibson assembly was evaluated by downstream experiments.

Agarose Gel Electrophoresis of PCR for 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

No.	Cloning intermediate
25	210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E)
26	250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP
27	260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP
28	280_pcDNAT7v1_RS20-cpEFGP-CaM
29	290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)
30	310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Protocol:

Agarose gel electrophoresis was performed using a 1.0% w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples (2 µL of sample with 3 µL of H₂O) mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 2 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (#Geldoc_model).

Results:

The following samples were loaded into the gel from left to right: NEB 1 kB ladder, 200_pcDNAT7v1, PCR20–PCR24, NEB 1 kB ladder, PCR25–PCR30. Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

Sanger sequencing of 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

To confirm the nucleotide sequences of 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4, Sanger sequencing was performed using Genewiz, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30–100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis did not find a match between the experimental and expected sequences.

Sample	Barcode	Primer	Validation
210.1	06817406	T7	No insert
210.1	06817414	T7-Term	No insert
250.1	06817405	T7	No insert
250.1	06817415	T7-Term	No insert
250.2	06817404	T7	No insert
250.2	06817416	T7-Term	No insert
250.3	06817407	T7	No insert
250.3	06817417	T7-Term	No insert
260.1	06817408	T7	No insert
260.1	06817418	T7-Term	No insert
260.2	06817409	T7	No insert
260.2	06817419	T7-Term	No insert
260.3	06817410	T7	No insert
260.3	06817420	T7-Term	No insert
280.1	06817411	T7	No insert
280.1	06817421	T7-Term	No insert
290.1	06817412	T7	No insert
290.1	06817422	T7-Term	No insert
310.1	06817413	T7	No insert
310.1	06817423	T7-Term	No insert

Agarose Gel Electrophoresis: gBlock @150, @151, @152, @153, @154, @155

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 based on their size.

Sample number	14	15	16	12	13	17
Primer	148 & 149	148 & 149	148 & 149	148 & 149	148 & 149	148 & 149
Fragment	152	153	154	150	151	155
Construct	260	280	290	210	250	310

Protocol:

Agarose gel electrophoresis was performed using a 1% w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5–10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right: L - NW - PCR12 - PCR13 - PCR14 - PCR15 - PCR16 - PCR17Overall, this experiment has proven some bands at the correct size, but unfortunately, most were smudged.

PCR (Q5® High-Fidelity DNA Polymerase) 12-17

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from the gBlocks @150, @151, @152, @153, @154, and @155 for constructs 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 with high fidelity and efficiency.

Constructs;

Sample number	14	15	16	12	13	17
Primer	148 & 149	148 & 149	148 & 149	148 & 149	148 & 149	148 & 149
kb	2.5	1.3	1.5	1	3	0.7
Fragment	152	153	154	150	151	155
Construct	260	280	290	210	250	310

Protocol:

PCR was performed using Q5® High-Fidelity DNA Polymerase (NEB), following manufacturer's instructions. For this, 10 ng DNA template, 12.5 pmol of forward ( 148_F_gBlock-SacI ) and reverse primer ( 149_R_gBlock-NcoI ) (final concentration 0.5 μM), and 12.5 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (#Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	25-μL run
Q5 High-Fidelity 2X Master Mix	1X	12.5 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2.5 μL
Template DNA	10 ng	2 μL
Nuclease-free water	–	8 μL

The reaction mixture was subjected to thermal cycling (#Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	5–10 sec	35
Annealing	62°C	10–30 sec
Extension	72°C	45 sec (12, 15, 16, 17) 1:15 min (13 & 14)
Final extension	72°C	2 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

PCR Agarose Gel Electrophoresis of the 12 - 17 samples

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP	280_pcDNAT7v1_RS20-cpEFGP-CaM	290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)	210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E)	250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP	310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4
14	15	16	12	13	17

Protocol:

Agarose gel electrophoresis was performed using a 1 % w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:6; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 6 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 20 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right: ladder - 12- 13 - 14 - 15 - 16 - 17

Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

PCR (Platinum™ SuperFi™ II)

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from the gBlocks @150, @151, @152, @153, @154, and @155 for constructs 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), @150, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 with high fidelity and efficiency.

Constructs:

12	13	14	15	16	17
150	151	152	153	154	155

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 20 ng DNA template, 10 pmol of each forward ( 148_F_gBlock-SacI ) and reverse ( 149_R_gBlock-NcoI ) primers (final concentration 0.5 μM), and 12.5 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	12.5 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2.5 μL
Template DNA	20 ng	2 μL
Nuclease-free water	–	8 μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. No heated lid was used.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	5–10 sec	25–35
Annealing	62°C	15 sec
Extension	72°C	35 sec (12, 17) 50 sec (16) 1 min 20 sec (14, 15) 1 min 35 sec (13)
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

Agarose Gel Electrophoresis - gBlocks @150, @151, @152, @153, @154, @155 with 148_F_gBlock-SacI & 149_R_gBlock-NcoI + 200_pcDNAT7v1

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Protocol:

Agarose gel electrophoresis was performed using a 1 % w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5–10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right: ladder - Q5 MM - 12 - 13 - 14 - 15 - 16 - 17 - NW - 200_pcDNAT7v1 digest_1 - 200_pcDNAT7v1 digest_2

Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples:

• No bands for inserts
• bands for backbone digestion at 3 kb instead of expected 6 kb

E. coli transformation with Gibson assembly

Goal:

Transformation was performed to introduce plasmid DNA construct 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, @190, and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

An aliquot of (new) competent DH5α  E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 µL of room temperature LB, 200 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. If a selection other than ampicillin was used, the mixture was first recovered at 37 °C with shaking (#Heatblock) prior to plating.

Results:

PCR (Q5® High-Fidelity DNA Polymerase) 12-17

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from gBlocks @150, @151, @152, @153, @154, and @155 for constructs 12, 13, 14, 15, 16, 17 & 18 with high fidelity and efficiency.

Protocol:

PCR was performed using Q5® High-Fidelity DNA Polymerase (NEB), following manufacturer's instructions. For this, 10 ng DNA template, 12.5 pmol of each forward and reverse primers (final concentration 0.5 μM), and 12.5 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	25-μL run
Q5 High-Fidelity 2X Master Mix	1X	12.5 μL
Forward + Reverse Primers (pre-mixed at 5 μM each) - 148_F_gBlock-SacI & 149_R_gBlock-NcoI	0.5 μM	2.5 μL
Template DNA	10 ng	2 μL
Nuclease-free water	–	8 μL

The reaction mixture was subjected to thermal cycling (#Cycler_model), which included 35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. In regards to extension time, we added 5 sec on top of the suggested 30 sec/kb to be sure that the fragments get extended correctly & fully.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	5–10 sec	25–35
Annealing	64°C	10–30 sec
Extension	72°C	35 sec (12, 17)50 sec (16)(15)1:20 min (13, 14)
Final extension	72°C	2 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

Agarose Gel Electrophoresis, PCR 12-17

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Plasmid name	260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP	280_pcDNAT7v1_RS20-cpEFGP-CaM	290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)	210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E)	250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP	310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4
Sample number	14	15	16	12	13	17

Protocol:

Agarose gel electrophoresis was performed using a 1% w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5–10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (#Geldoc_model).

Results:

The following samples were loaded into the gel from left to right: ladder - 17 - 16 - 15 - 14 - 13 - 12 - ladder

Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples (so none).

Agarose Gel Electrophoresis of analytical digestion of 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4, 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples- 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4, 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES based on their size.

Protocol:

Agarose gel electrophoresis was performed using a 1% w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5–10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right: DNA ladder, 280.3, 290.3, 310.1, 590.1, 600.1, 610.2.,water, DNA ladder Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples 590.1, 600.1 and 610.2. No bands of corresponding mass was seen for constructs 280.3, 290.3, 310.1. Additionally DNA ladder did not function properly, showing no sharp bands.

Clean-up (NEB) of digested backbone 200_pcDNAT7v1

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (#TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL Eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of 2x10 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (#Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
200_pcDNAT7v1 digest_1	1.59	0.79	-0.13
200_pcDNAT7v1 digest_2	0.72	1.28	-0.06

Agarose Gel Electrophoresis 330_LET_CLS-XaCS-BmTyr, 340_LET_CLS-XaCS-BmTyr(E195S+A221V), 350_LET_CLS-XaCS-BmTyr(R209S+M215E+V218M), 360_LET_CLS-XaCS-BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C), 370_LET_CLS-XaCS-cpBmTyr(201), 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201), 440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466), 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M), 460_LET_CLS-XaCS-LsTyr, 470_LET_CLS-XaCS-SkMelC1(33-124), 480_LET_CLS-XaCS-SkMelC2, 490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518), 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M), 510_LET_CLS-XaCS-SavMelC1(27-118), 520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F), 530_LET_CLS-XaCS-SavMelC2, 540_LET_CLS-XaCS-SavMelC2(I42Y)

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples- 330_LET_CLS-XaCS-BmTyr, 340_LET_CLS-XaCS-BmTyr(E195S+A221V), 350_LET_CLS-XaCS-BmTyr(R209S+M215E+V218M), 360_LET_CLS-XaCS-BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C), 370_LET_CLS-XaCS-cpBmTyr(201), 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201), 440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466), 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M), 460_LET_CLS-XaCS-LsTyr, 470_LET_CLS-XaCS-SkMelC1(33-124), 480_LET_CLS-XaCS-SkMelC2, 490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518), 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M), 510_LET_CLS-XaCS-SavMelC1(27-118), 520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F), 530_LET_CLS-XaCS-SavMelC2, 540_LET_CLS-XaCS-SavMelC2(I42Y) based on their size.

Protocol:

Agarose gel electrophoresis was performed using a 1 % w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5–10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right:

Gel 1: Ladder, Ladder, 330_LET_CLS-XaCS-BmTyr, 340_LET_CLS-XaCS-BmTyr(E195S+A221V), 350_LET_CLS-XaCS-BmTyr(R209S+M215E+V218M), 360_LET_CLS-XaCS-BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C), 370_LET_CLS-XaCS-cpBmTyr(201), 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201), 440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466)

Gel 2: Ladder, Ladder, 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M), 460_LET_CLS-XaCS-LsTyr, 470_LET_CLS-XaCS-SkMelC1(33-124), 490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518), 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M), 510_LET_CLS-XaCS-SavMelC1(27-118), 480, 520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F), 530_LET_CLS-XaCS-SavMelC2, 540_LET_CLS-XaCS-SavMelC2(I42Y), 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M) GA, 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M) GA

Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

E. coli transformation with GA of 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, @190, and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

git config pull.rebase false

Goal:

Transformation was performed to introduce plasmid DNA construct 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

An aliquot of competent DH5α  E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (2 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 µL of room temperature SOC, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. If a selection other than ampicillin was used, the mixture was first recovered at 37 °C with shaking (#Heatblock) prior to plating.

Results:

Gibson Assembly of constructs 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), @150, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

Gibson assembly was performed to assemble the 200_pcDNAT7v1 vector and @150, @151, @152, @153, @154, and @155 inserts to produce 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), @150, @160, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4.

Protocol:

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Components	Volume
NEBuilder HiFi DNA Assembly Master Mix	10 µL
Vector	1 ng
Insert	3 ng
Nuclease-free Water	6 μL
Total	20 µL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of Gibson assembly was evaluated by downstream experiments.

PCR (Q5® High-Fidelity DNA Polymerase) 1-11

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from @150, @151, @152, @153, @154, and @155, 200_pcDNAT7v1 for constructs 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 with high fidelity and efficiency.

Constructs:

	1	2	3	4	5	6	7	8	9	10	11
Primer	251_F_SacI-Kozak-CD4SP_T7p & 252_R_BFP-Stop-NcoI_T7tt	253_F_NcoI-T7tt_BFP-Stop & 254_R_T7p-SacI_Kozak-CD4SP	281_F_SacI-RS20_T7p & 282_R_CaM-NcoI_pcDNA	283_F_NcoI-pcDNA_CaM & 254_R_T7p-SacI_Kozak-CD4SP	281_F_SacI-RS20_T7p & 292_R_CTEVp-Stop-NcoI_T7tt	293_F_NcoI-T7tt_CTEVp-Stop & 254_R_T7p-SacI_Kozak-CD4SP	251_F_SacI-Kozak-CD4SP_T7p & 212_R_NTEVp-Stop-NcoI_T7tt	251_F_SacI-Kozak-CD4SP_T7p & 252_R_BFP-Stop-NcoI_T7tt	213_F_NcoI-T7tt_NTEVp-Stop & 254_R_T7p-SacI_Kozak-CD4SP	281_F_SacI-RS20_T7p & 312_R_P4-Stop-NcoI_T7tt	313_F_NcoI-T7tt_P4-Stop & 254_R_T7p-SacI_Kozak-CD4SP
kb	2.5	6	1.3	6	1.5		1	3	6	0.7	6
Fragment	152	200	153	200	154	200	150	151	200	155	200
Construct	260	260, 250	280	280	290	290	210	250	210	310	310

Protocol:

PCR was performed using Q5® High-Fidelity DNA Polymerase (NEB), following manufacturer's instructions. For this, 20 ng DNA template, 12.5 pmol of each forward and reverse primers (final concentration 0.5 μM), and 12.5 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	25-μL run
Q5 High-Fidelity 2X Master Mix	1X	12.5 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2.5 μL
Template DNA	0.8 ng/µL	2 μL
Nuclease-free water	–	8 μL

The reaction mixture was subjected to thermal cycling (#Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	5–10 sec 2 min (2, 4, 6, 9, 11)	25–35
Annealing	63°C (1, 8) 62°C (2, 4, 6, 9, 11) 63°C (3,7) 60 °C (5, 10)	10–30 sec
Extension	72°C	1:30 min (1, 8) 3:30 min (2, 4, 6, 9, 11) 0:45 min (3,7) 0:45 min (5, 10)
Final extension	72°C	2 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments. PCRs 2, 4, 6, 9, 11 were wrongly programmed and need to be redone.

gBlocks resuspension

Goal:

The gBlocks @150, @151, @152, @153, and @155 from TWIST Bioscience and the gBlock @154 from IDT were resuspended in IDTE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) to a final concentration of 10 ng/μL.

Protocol:

Lyophilized gBlocks were briefly centrifuged to collect the pellet at the bottom of the tube. The DNA was resuspended by vortexing in nuclease-free IDTE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) to reach the desired concentration of 10 ng/μL.

Results:

The resuspended gBlocks were stored at −20 °C and used for subsequent downstream experiments.

PCR (Q5® High-Fidelity DNA Polymerase) - 1-11 hotstart method

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from @150, @151, @152, @153, @154, @155 and 200_pcDNAT7v1 for constructs 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP,260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 with high fidelity and efficiency.

Protocol:

PCR was performed using Q5® High-Fidelity DNA Polymerase (NEB), following manufacturer's instructions. For this, 10 ng DNA template, 12.5 pmol of each forward and reverse primers (final concentration 0.5 μM), and 12.5 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	25-μL run
Q5 High-Fidelity 2X Master Mix	1X	12.5 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2.5 μL
Template DNA	10 ng	2 μL
Nuclease-free water	–	8 μL

In regard of the construct and primer combination, we used the same combinations as in the PCR before, as this was a repeat. The reaction was pipetted on ice and placed in the already warmed-up cycler, making this a Hotstart PCR.

The reaction mixture was subjected to thermal cycling (#Cycler_model), which included 30 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec2 min (2, 4, 6, 9, 11)	1
Denaturation	98°C	10 sec15 sec (2, 4, 6, 9, 11)	30
Annealing	62°C	15 sec
Extension	72°C	1:30 min (1, 8) 3:30 min (2, 4, 6, 9, 11) 0:45 min (3,7) 4:30 min
Final extension	72°C	2 min10 min (2, 4, 6, 9, 11)	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

PCR (Platinum™ SuperFi™ II) of 200_pcDNAT7v1 and @154

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments for constructs 200_pcDNAT7v1 and @154 with high fidelity and efficiency.

Part	Primers	Length	Extension time
200	293 & 254	6	3 min 15 sec
154	281 & 292	1.5	1 min

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA	10 ng	1 μL
DMSO		0.5 µL
Nuclease-free water	–	6.5 μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	10 sec	25
Annealing	58°C	20 sec
Extension	72°C	30 seconds/kb
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

PCR (Q5® High-Fidelity DNA Polymerase) - 2, 4, 6, 9, 11

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 200_pcDNAT7v1 for constructs 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 with high fidelity and efficiency.

Protocol:

PCR was performed using Q5® High-Fidelity DNA Polymerase (NEB), following manufacturer's instructions. For this, 10 ng DNA template, 12.5 pmol of each forward and reverse primers (final concentration 0.5 μM), and 12.5 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	25-μL run
Q5 High-Fidelity 2X Master Mix	1X	12.5 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2.5 μL
Template DNA	0.8 ng	2 μL
Nuclease-free water	–	8 μL

The reaction mixture was subjected to thermal cycling (#Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	2 min	25–35
Annealing	62°C	10–30 sec
Extension	72°C	3:30 sec/kb
Final extension	72°C	10 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

Agarose Gel Electrophoresis - inserts 1,3, 5, 7, 8, 10

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Protocol:

Agarose gel electrophoresis was performed using a 1% w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA: 450ml 10x clean water + 50 ml 10x TAE) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5–10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 20 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right:Ladder - NW (nuclease free water) - Q5MM - Template ( 200_pcDNAT7v1 ) - PCR1 - PCR3 - PCR5 - PCR7 - PCR8 - PCR10Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples, except for PCR10.

Restriction digest of 320_pcDNAT7v2

Goal:

Restriction digest of the 320.

Protocol:

Restriction digest was carried out using an enzyme(s)  (New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 3 hour.

Component	Volume
Restriction enzyme	50 U (5 μL)
rCutSmart buffer	15 μL
DNA	5 μg
Nuclease-free Water	to 50 μL

The products were then stored at –20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

Plasmid Miniprep (NEB) of 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal:

Minipreps of 290 were performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol:

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight cultures were pelleted by centrifugation for 15 minutes at 4000 ×  g (Eppendorf, Centrifuge 5804R). For each sample, the supernatant was discarded. Pellet was resuspended in 200 μL of plasmid resuspension buffer. Cell lysis was achieved by the addition of 200 μL of plasmid lysis buffer, followed by 5–6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of plasmid neutralization buffer. The tubes were gently inverted and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transferred to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of plasmid wash buffer 1 and centrifuging for 1 minute, followed by 400 μL of plasmid wash buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL Eppendorf tubes. DNA was eluted by the addition of 30 μL of preheated TE buffer (55–65 °C) to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop (#Nano_model), and the DNA samples were stored at −20 °C.

Results:

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
290.1	323.77	1.85	2.53
290.2	551.18	1.86	2.29
290.3	517.33	1.88	2.40
290.4	394.79	1.86	2.47

Agarose Gel Electrophoresis of digested 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), @172 and @173

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples 290, 172 and 173 based on their size.

Protocol:

Agarose gel electrophoresis was performed using a ...% w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5–10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right:

L-290.1-290.2-290.3-290.4-L-172-173

Overall, the agarose gel electrophoresis analysis may have confirmed the presence and size distribution of the expected DNA fragments in the samples.

Restriction digest of @172 and @173

Goal:

Restriction digest of the @172 and @173 with KpnI and HindIII enzyme(s), for downstream cloning of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES.

Protocol:

Restriction digest was carried out using the KpnI and HindIII enzyme(s) (…,  New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 1 hour.

Component	Volume
KpnI	1 U (0.1 μL)
HindIII	1 U (0.1 μL)
rCutSmart buffer	5 μL
DNA	1 μg
Nuclease-free Water	to 10 μL

Optionally, the reaction was stopped using heat inactivation (80 °C, 20 minutes). The products were then stored at –20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

Plasmid Maxiprep (QIAGEN) of 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal:

A maxiprep of 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Protocol:

DNA plasmids were prepared using the QIAGEN ® Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). 200 mL of overnight culture of E. coli was centrifuged at 4500 × g for 20 min at 4°C. The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer with thorough mixing. The mixture was centrifuged at 7000 × g (eppendorf 5430 R) for 135 min (if supernatant not clear, optional +30) min at 4 °C. The supernatant was applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuging at 7000 × g for 75 min (if pellet not strong enough, additional 30 min) at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol. Finally, the supernatant was aspirated using a vacuum pump without disturbing the pellet, and the pellet was air-dried for 5 - 10 min. The DNA was then redissolved in 200 μL of TE buffer, the concentration was determined using a NanoDrop, and the DNA was stored at −20 °C.

Results:

The maxiprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
290	1796	1.9	2.2

QIAGEN Maxiprep of 200_pcDNAT7v1

Things to do before starting:

• Before use, centrifuge RNase A briefly, and then add into Buffer P1 to obtain a final concentration of 100 μg/mL
• Check Buffer P2 for SDS precipitation due to low storage temperatures. If necessary, dissolve the SDS by warming to 37°C.
• Prechill Buffer P3 on ice.
• If not prepared: Add the provided LyseBlue reagent to Buffer P1 and mix before use. Use 1 vial LyseBlue reagent per bottle Buffer P1 for a final dilution of 1:1000 (e.g., 10 µL LyseBlue into 10 mL Buffer P1).

Maxi prep:

1. Harvest the bacterial cells of the culture with 200_pcDNAT7v1 by centrifugation at 6000 x g for 15 min at 4°C. (If you wish to stop the protocol and continue later, freeze the cell pellets at −30 to −15°C.)
2. Resuspend the bacterial pellet in 10 mL of Buffer P1. Ensure that RNase A has been added to Buffer P1, shake the buffer bottle before use. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
3. Add 10 mL of Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4−6 times, and incubate at room temperature for 5 min (not longer!). Do not vortex because this will result in shearing of genomic DNA! After use, the bottle containing Buffer P2 should be closed immediately to avoid acidification from CO2 in the air.
4. Add 10 ml prechilled Buffer P3, mix thoroughly by vigorously inverting 4–6 times. Incubate on ice for 20 min. If using LyseBlue reagent, mix the solution until it is colorless.
5. Centrifuge at ≥20,000 x g for 30 min at 4°C. Re-centrifuge the supernatant at ≥20,000 x g for 15 min at 4°C.
6. Equilibrate a QIAGEN-tip 500 by applying 10 ml Buffer QBT, and allow column to empty by gravity flow.
7. Apply the supernatant from step 5 to the QIAGEN-tip and allow it to enter the resin by gravity flow.
8. Wash the QIAGEN-tip with 2 x 30 ml Buffer QC. Allow Buffer QC to move through the QIAGEN-tip by gravity flow.
9. Elute DNA with 15 ml Buffer QF into a clean 50 ml vessel. For constructs larger than 45 kb, prewarming the elution buffer to 65°C may help to increase the yield.
10. Precipitate DNA by adding 10.5 ml (0.7 volumes) room temperature isopropanol to the eluted DNA and mix. Centrifuge at ≥15,000 x g for 2 h at 4°C. Carefully decant the supernatant. If possible, try to transfer the pellet into a 2 mL Eppendorf
11. Wash the DNA pellet with 5 ml room-temperature 70% ethanol and centrifuge at ≥15,000 x g for 10 min. Carefully decant supernatant. If you have your pellet in a 2 mL Eppendorf, wash twice with 2 mL 70% ethanol.
12. Air-dry pellet for 5–10 min and redissolve DNA in a suitable volume of appropriate buffer (e.g., TE buffer, pH 8.0, or 10 mM Tris·Cl, pH 8.5). Start with 200 µL buffer, measure the concentration with the Nanodrop, and add more buffer volume if necessary. Write down the plasmid concentration and the ratios 260/280 and 260/230.
13. Make a 200 µL working stock solution with a plasmid concentration of 100 ng/µL
14. Store the working stock solution in the fridge and the original stock solution in the freezer

Results:

Plasmid name	Concentration, ng/µL	260/280	260/230
200_pcDNAT7v1 _1	2574,84	1,96	2,25
200_pcDNAT7v1 _2	2574,84	1,94	2,22

Sanger sequencing of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP and 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal:

To confirm the nucleotide sequences of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP and 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a (partial) match between the experimental and expected sequences. In addition, the sequencing data showed (predominantly) a high quality score and a continuous read length of over 1 kb.

Sample	Barcode	Primer	Validation
270.7	06817623	CMV	template 250
270.7	06817624	WPRE	template 250
270.8	06817625	CMV	insert mutation
270.8	06817626	WPRE	insert mutation
270.9	06817627	CMV	mutations
270.9	06817628	WPRE	mutations
290.2	06817629	CMV	validated
290.2	06817630	WPRE	validated
290.3	06817631	CMV	many mutations
290.3	06817632	WPRE	many mutations

DpnI digest of 320_pcDNAT7v2 for 630_pcDNAT7v2_iCasp9-T2A-GFP

Goal:

Restriction digest of enzyme(s), for downstream cloning of 320_pcDNAT7v2 for 630_pcDNAT7v2_iCasp9-T2A-GFP.

Protocol:

Restriction digest was carried out using an enzyme(s) (New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 1 hour.

Component	Volume
Restriction enzyme	10 U (1 μL)
rCutSmart buffer	5 μL
DNA	1 μg
Nuclease-free Water	to 50 μL

Optionally, the reaction was stopped using heat inactivation (80 °C, 20 minutes). The products were then stored at –20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

DpnI digest of 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) to generate 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal:

Restriction digest of the amplified 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) with DpnI enzymes, for downstream cloning of 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K).

Protocol:

Restriction digest was carried out using an enzyme(s) ( New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 1 hour.

Component	Volume
Restriction enzyme	10 U (1 μL)
rCutSmart buffer	5 μL
DNA	1 μg (7.4 µL)
Nuclease-free Water	to 50 μL

Optionally, the reaction was stopped using heat inactivation (80 °C, 10 minutes). The products were then stored at –20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

PCR (Platinum™ SuperFi™ II) - Tyrosinases

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from ... for constructs ... with high fidelity and efficiency.

	Construct	Template	Primer	length	extension time
1	660	pET	661 & 664	5.4 kb	2 min 20 sec
2	177	663 & 662	105 bp	10 sec
3	670	157	671 & 672	891 bp	40 sec
4	680	158	671 & 672	891 bp	40 sec
5	690	160	671 & 672	891 bp	40 sec
6	700	159	671 & 672	891 bp	40 sec
7	710	161	711 & 712	903 bp	40 sec
8	721	157	671 & 722	255 bp	20 sec
9	731	157	732 & 672	639 bp	30 sec
10	741	157	671 & 742	471 bp	30 sec
11	751	157	752 & 672	423 bp	30 sec
12	761	157	671 & 762	603 bp	30 sec
13	771	157	772 & 672	291 bp	20 sec
14	790	162	791 & 792	1.3 kb	1 min
15	810	162	811 & 791	909 bp	40 sec
16	820	163	822 & 821	942 bp	40 sec
17	830	167	831 & 832	279 bp	20 sec
18	840	169	842 & 841	822 bp	40 sec
19	850	168	831 & 832	279 bp	20 sec
20	860	170	842 & 841	822 bp	40 sec
21	870	164	871 & 872	279 bp	20 sec
22	880	165	881 & 882	819 bp	40 sec
23	890	166	891 & 892	1.5 kb	1 min
24	900	166	891 & 901	996 bp	40 sec

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA	10 ng	X μL
DMSO		0.5 µL
Nuclease-free water	–	X μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	5–10 sec	25
Annealing	60°C	20 sec
Extension	72°C	30 seconds/kb
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

PCR Clean-up (NEB) of @172 and @173

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g. The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g. DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Restriction digest of @172 and @173

Goal:

Restriction digest of the with enzyme(s), for downstream cloning of @172 and @173.

Protocol:

Restriction digest was carried out using a enzyme(s)  (New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 1 hour.

Component	Volume
Restriction enzyme	10 U (1 μL)
rCutSmart buffer	5 μL
DNA	1 μg
Nuclease-free Water	to 50 μL

Optionally, the reaction was stopped using heat inactivation (80 °C, 20 minutes). The products were then stored at –20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

Analytical digest of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Restriction digest of enzyme(s) for downstream cloning of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES.

Protocol:

Restriction digest was carried out using the enzyme(s) (New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 1 hour.

The mastermix was pipetted as follows:

Component	Volume
KpnI	0.9 μL
HindIII	0.9
rCutSmart buffer	0.5 μL
Nuclease-free Water	81.9 μL

9.8 µL mastermix was added to 0.2 µL of DNA.

Optionally, the reaction was stopped using heat inactivation (80 °C, 10 minutes). The products were then stored at –20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

PCR Clean-up (NEB) of 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

PCR Clean-up (NEB) of digested @172 and @173

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

PCR (Platinum™ SuperFi™ II) of 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) to generate 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)  for construct 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K) with high fidelity and efficiency.

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)221 & 222	0.5 μM	2 μL
Template DNA	10 ng	X μL
Nuclease-free water	–	X μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	5–10 sec	25–35
Annealing	60°C	10 sec
Extension	72°C	4:30 min
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

PCR Clean-up (NEB) of amplified and DpnI digested 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) to generate 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Analytical digest of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Restriction digest of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES with KpnI-HF and HindIII-HF enzymes.

Protocol:

Restriction digest was carried out using the enzyme(s) ( New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 1 hour.

The mastermix was pipetted as follows:

Component	Volume
KpnI	3.3 μL
HindIII	3.3 µL
rCutSmart buffer	18.7 μL
Nuclease-free Water	73.7 μL

9 µL mastermix was added to 1 µL of DNA.

Optionally, the reaction was stopped using heat inactivation (80 °C, 10 minutes). The products were then stored at –20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

Amplification of DNA using the phi29-XT RCA Kit for purified circular DNA for 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

DNA of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES was amplified using the phii29-XT RCA kit (NEB #E1603) for downstream applications.

Protocol:

Reactions with an end volume of 20 µL were prepared without enzyme as described in the table below. For each reaction, 4 µL of phi29-XT Reaction Buffer (5X) was added to achieve a final concentration of 1X, as well as 2 µL of Deoxynucleotide (dNTP) Solution Mix (10 mM) for a final concentration of 1 mM.  Furthermore, 2 µL of Exonuclease-Resistant Random Primers (500 µM) were added to achieve a final concentration of 50 µM, as well as X µL of the  DNA template for a final concentration of X. Nuclease-free water was used to bring the reaction to 18 µL.

Components	20 µL reaction	Final concentration
phi29-XT Reaction Buffer, 5X	4 µL	1X
Deoxynucleotide (dNTP) Solution Mix, 10 mM	2 µL	1 mM
Exonuclease-Resistant Random Primers, 500 µM	2 µL	50 µM
Circular DNA Template	10 µL	variable
Nuclease-free Water	to 18 µL	N/A

Each reaction was mixed thoroughly by gentle pipetting or vortexing, followed by brief centrifuging.

After that, the reactions were incubated in a thermocycler (model) at 95 °C for 3 minutes, with a lid temperature of 100 °C, followed by cooling the reactions at room temperature.

The reactions were put on ice, and 2 µL of phi29-XT DNA Polymerase was added to each reaction, followed by mixing by gentle pipetting or vortexing, and brief centrifuging.

Lastly, the reactions were incubated in a thermocycler (model) at 42 °C for 2 hours, with a lid temperature of ≥ 75°C, followed by 10 minutes at 65 °C to inactivate the DNA polymerase.

The RCA products can be kept at 4 °C overnight or at  -20 °C for long-term storage.

Results:

KLD of 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal:

KLD reaction was performed to assemble constructs 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K) from PCR products .

Protocol:

KLD reaction was performed using the KLD Enzyme Mix (M0554, New England Biolabs), consisting of a kinase, a ligase, and a DpnI restriction enzyme, according to the manufacturer's instructions. For each reaction, KLD reaction buffer, KLD enzyme mix, nuclease-free water, and the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 μL
KLD Reaction Buffer (2x)	5 μL
KLD Enzyme Mix (10x)	1 μL
Nuclease-free water	3 μL
Total	10 μL

The sample was incubated at room temperature (25 °C) for 5–10 minutes. Optionally, to increase the efficiency of the DpnI digestion, the mixture was additionally incubated at 37 °C for 30–60 minutes. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of KLD reaction was evaluated by downstream experiments.

Analytical digest of RCA with 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Restriction digest of enzyme(s), for downstream cloning of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Protocol:

Restriction digest was carried out using the  enzyme(s) (New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 1 hour.

Component	Volume
KpnI	1 μL
HindIII	1 µL
rCutSmart buffer	5 μL
DNA	1 μL
Nuclease-free Water	to 50 μL

Optionally, the reaction was stopped using heat inactivation (80 °C, 20 minutes). The products were then stored at –20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

E. coli transformation 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES, 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K) and Re-trafo of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP

Goal:

Transformation was performed to introduce plasmid DNA constructs 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES, 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K) and Re-trafo of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP.

into DH10β E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

For each construct, an aliquot of competent DH10β E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells (1–5 μL) and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 μL of room temperature SOC, the mixture was then recovered for 30–60 min at 37 °C with shaking (#Heatblock). The mixture was centrifuged at 16000 x g for 30 seconds to remove the supernatant. The pellet was resuspended in 50 μl of LB and spread onto a selection plate and incubated overnight at 37°C.

Results:

Gibson Assembly of 630_pcDNAT7v2_iCasp9-T2A-GFP

Goal:

Gibson assembly was performed  to produce 630_pcDNAT7v2_iCasp9-T2A-GFP

Protocol:

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Components	Volume
NEBuilder HiFi DNA Assembly Master Mix	10 µL	10 µL
Vector (320_pcDNAT7v2)	50 ng	1.5 µL
Insert (@175)	150 ng	1.5 µL
Insert (@176)	150 ng	0.8 µL
Nuclease-free Water	... μL	6.2 µL
Total	20 µL	20 µL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of Gibson assembly was evaluated by downstream experiments.

E. coli transformation with 570_pAAVS1_CAGp-HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)_IRES-BSD, 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES, 630_pcDNAT7v2_iCasp9-T2A-GFP, and 660_pET_21(+)_Twin-Strep

Goal:

Transformation was performed to introduce plasmid DNA constructs 570_pAAVS1_CAGp-HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)_IRES-BSD, 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES, 630_pcDNAT7v2_iCasp9-T2A-GFP, and 660_pET_21(+)_Twin-Strep  into DH10β E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

For each construct, an aliquot of competent DH10β E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells (1–5 μL) and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 μL of room temperature SOC, the mixture was then recovered for 30–60 min at 37 °C with shaking (#Heatblock). The mixture was centrifuged at 16000 x g for 30 seconds to remove the supernatant. The pellet was resuspended in 50 μl of LB and spread onto a selection plate and incubated overnight at 37°C.

Results:

Gibson Assembly of 660_pET_21(+)_Twin-Strep and 570_pAAVS1_CAGp-HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)_IRES-BSD

Goal:

Gibson assembly was performed to produce 660_pET_21(+)_Twin-Strep and 570_pAAVS1_CAGp-HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)_IRES-BSD.

Protocol:

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Components	Volume	660_pET_21(+)_Twin-Strep	570_pAAVS1_CAGp-HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)_IRES-BSD
NEBuilder HiFi DNA Assembly Master Mix	10 µL	10 µL	10 µL
Vector	50 ng	1.3 µL	0.9 µL
Insert	100 ng	1.7 µL	0.6 µL
Nuclease-free Water	X	7 µL	8.5 µL
Total	20 µL	20 µL	20µL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of Gibson assembly was evaluated by downstream experiments.

ZymoPURE™ Plasmid Miniprep Kit for 570_pAAVS1_CAGp-HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)_IRES-BSD, 630_pcDNAT7v2_iCasp9-T2A-GFP, and 660_pET_21(+)_Twin-Strep

Goal:

Purify plasmid DNA without endotoxins.

Protocol:

The Minipreps were done following the manufacturer's (ZYMOPURE) protocol.

Before starting, make sure that ethanol was added to the ZymoPURE Wash 2 buffer (88 ml of 95 – 100% ethanol to the 23 ml ZymoPURE™ Wash 2 (Concentrate) (D4210)). The ZymoPURE™ P2 and ZymoPURE™ Binding Buffer may have precipitated.  If this occurs, dissolve the precipitate by incubating the bottles at 30-37 ºC for 10-20 minutes and mix by inversion.  Do not microwave!

E. coli colony picking of 570_pAAVS1_CAGp-HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)_IRES-BSD, 630_pcDNAT7v2_iCasp9-T2A-GFP, and 660_pET_21(+)_Twin-Strep

Goal

Colony picking of individual DH10β E. coli (Cat. No. C3019, New England Biolabs) bacterial colonies transformed with 570_pAAVS1_CAGp-HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)_IRES-BSD, 630_pcDNAT7v2_iCasp9-T2A-GFP, and 660_pET_21(+)_Twin-Strep from selection plates containing carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plates. Each colony was transferred to 5 mL of fresh culture medium (LB medium with 100 μg/mL carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm (CERTOMAT BS-1 Incubator Shaker, Sartorius) for further growth and amplification of the selected cells. The agar plates were sealed to prevent contamination and stored at 4 °C.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial cultures after overnight incubation at 37 °C.

Agarose Gel Electrophoresis of 630_pcDNAT7v2_iCasp9-T2A-GFP and 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Protocol:

Agarose gel electrophoresis was performed using a  1%  agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5–10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right:

L-630.1-630.2-630.3-640.4-630.5-L-300.1-300.3-300.4

Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

E. coli transformation with 560_pAAVS1_CAGp-IL7Ra-IRES-BSD and 630_pcDNAT7v2_iCasp9-T2A-GFP

Goal:

Transformation was performed to introduce plasmid DNA constructs 560_pAAVS1_CAGp-IL7Ra-IRES-BSD and 630_pcDNAT7v2_iCasp9-T2A-GFP into DH10β E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

For each construct, an aliquot of competent DH10β E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells (1–5 μL) and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 μL of room temperature SOC, the mixture was then recovered for 30–60 min at 37 °C with shaking (#Heatblock). The mixture was centrifuged at 16000 x g for 30 seconds to remove the supernatant. The pellet was resuspended in 50 μl of LB and spread onto a selection plate and incubated overnight at 37°C.

Results:

Analytical digest of 630_pcDNAT7v2_iCasp9-T2A-GFP and 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal:

Restriction digest for downstream cloning of 630_pcDNAT7v2_iCasp9-T2A-GFP and 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K)

Protocol:

Restriction digest was carried out using the enzymes ( New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 1 hour.

The mastermix for 630_pcDNAT7v2_iCasp9-T2A-GFP was pipetted as follows:

Component	Volume
KpnI	3 μL
HindIII	3 µL
rCutSmart buffer	15 μL
Nuclease-free Water	33 μL

9 µL of mastermix was added to 1 µL of DNA (1:10 diluted from RCA).

The mastermix for 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K) was pipetted as follows:

Component	Volume
KpnI	0.8 μL
HindIII	0.8 µL
rCutSmart buffer	4 μL
Nuclease-free Water	30 μL

9 µL of mastermix was added to 1 µL of DNA.

Optionally, the reaction was stopped using heat inactivation (80 °C, 20 minutes). The products were then stored at –20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

E. coli colony picking of 630_pcDNAT7v2_iCasp9-T2A-GFP

Goal

Colony picking of individual DH10β E. coli (Cat. No. C3019, New England Biolabs) bacterial colonies transformed with 630_pcDNAT7v2_iCasp9-T2A-GFP from selection plates containing carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plates. Each colony was transferred to 5 mL of fresh culture medium (LB medium with 100 μg/mL carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm (CERTOMAT BS-1 Incubator Shaker, Sartorius) for further growth and amplification of the selected cells. The agar plates were sealed to prevent contamination and stored at 4 °C.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial cultures after overnight incubation at 37 °C.

E. coli transformation of pET21b

Goal:

Transformation was performed to introduce plasmid DNA pET21b into DH10β E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

For each construct, an aliquot of competent DH10β E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells (1–5 μL) and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 μL of room temperature SOC, the mixture was then recovered for 30–60 min at 37 °C with shaking (#Heatblock). The mixture was centrifuged at 16000 x g for 30 seconds to remove the supernatant. The pellet was resuspended in 50 μl of LB and spread onto a selection plate and incubated overnight at 37°C.

Results:

E. coli transformation of 660_pET_21(+)_Twin-Strep, 910_pcDNAT7v2_BmTyr-TEVCS(M)-MxEnc-eUnaG-STII-NES, 920_pcDNAT7v2_BmTyr-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 930_pcDNAT7v2_BmTyr-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Transformation was performed to introduce plasmid DNA constructs 660_pET_21(+)_Twin-Strep, <q910, 920_pcDNAT7v2_BmTyr-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 930_pcDNAT7v2_BmTyr-TEVCS(M)-TmEnc-eUnaG-STII-NES into DH10β E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

For each construct, an aliquot of competent DH10β E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells (1–5 μL) and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 μL of room temperature SOC, the mixture was then recovered for 30–60 min at 37 °C with shaking (#Heatblock). The mixture was centrifuged at 16000 x g for 30 seconds to remove the supernatant. The pellet was resuspended in 50 μl of LB and spread onto a selection plate and incubated overnight at 37°C.

Results:

Gibson Assembly of 660_pET_21(+)_Twin-Strep, 910_pcDNAT7v2_BmTyr-TEVCS(M)-MxEnc-eUnaG-STII-NES, 920_pcDNAT7v2_BmTyr-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 930_pcDNAT7v2_BmTyr-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Gibson assembly was performed to assemble to produce 660_pET_21(+)_Twin-Strep, 910_pcDNAT7v2_BmTyr-TEVCS(M)-MxEnc-eUnaG-STII-NES, 920_pcDNAT7v2_BmTyr-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 930_pcDNAT7v2_BmTyr-TEVCS(M)-TmEnc-eUnaG-STII-NES

Protocol:

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Components	Volume
NEBuilder HiFi DNA Assembly Master Mix	10 µL
Vector	50 ng
Insert	30.41 ng
Nuclease-free Water	... μL
Total	20 µL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of Gibson assembly was evaluated by downstream experiments.

Gibson Assembly 630_pcDNAT7v2_iCasp9-T2A-GFP, 640_pcDNAT7v2_EGFP, 650_pcDNAT7v2_CD4SP-L-CD28-CS(G)-tTAEmma >

2025-09-10

Emma Schwarze

Goal:

Gibson assembly was performed  to produce 630_pcDNAT7v2_iCasp9-T2A-GFP, 640_pcDNAT7v2_EGFP, 650_pcDNAT7v2_CD4SP-L-CD28-CS(G)-tTAEmma>

Protocol:

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Components	Volume
NEBuilder HiFi DNA Assembly Master Mix	10 µL
Vector	50 ng
Insert	30 ng
Nuclease-free Water	... μL
Total	20 µL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of Gibson assembly was evaluated by downstream experiments.

DpnI digest of 320_pcDNAT7v2 for 640_pcDNAT7v2_EGFP and 650_pcDNAT7v2_CD4SP-L-CD28-CS(G)-tTAEmma>

2025-09-10

Emma Schwarze

Goal:

Restriction digest  for downstream cloning of 320_pcDNAT7v2 for 640_pcDNAT7v2_EGFP and 650_pcDNAT7v2_CD4SP-L-CD28-CS(G)-tTAEmma>

Protocol:

Restriction digest was carried out using enzyme (New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 1 hour.

Component	Volume
Restriction enzyme	10 U (1 μL)
rCutSmart buffer	5 μL
DNA	1 μg
Nuclease-free Water	to 50 μL

Optionally, the reaction was stopped using heat inactivation (80 °C, 20 minutes). The products were then stored at –20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

PCR (Platinum™ SuperFi™ II) of 630_pcDNAT7v2_iCasp9-T2A-GFP, 640_pcDNAT7v2_EGFP, and 650_pcDNAT7v2_CD4SP-L-CD28-CS(G)-tTAEmma>

2025-09-10

Emma Schwarze

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments for constructs 630_pcDNAT7v2_iCasp9-T2A-GFP, 640_pcDNAT7v2_EGFP, and 650_pcDNAT7v2_CD4SP-L-CD28-CS(G)-tTAEmma high fidelity and efficiency.

Composite part	Template	Primer	Length	Extension time
630	176	631 & 632	1.3	50 sec
640	320	321 & 322	6	3 min 15 sec
	175	641 & 642	1.2	50 sec
650	320	953 & 752	6	3 min 15 sec
	174	651 & 652	2.1	1 min 15 sec

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA	10 ng	X μL
Nuclease-free water	–	X μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	5–10 sec	25
Annealing	60°C	20 sec
Extension	72°C	30 seconds/kb
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

E. coli transformation with 630_pcDNAT7v2_iCasp9-T2A-GFP, 640_pcDNAT7v2_EGFP, 650_pcDNAT7v2_CD4SP-L-CD28-CS(G)-tTAEmma>

2025-09-10

Emma Schwarze

Goal:

Transformation was performed to introduce plasmid DNA constructs 630_pcDNAT7v2_iCasp9-T2A-GFP, 640_pcDNAT7v2_EGFP, 650_pcDNAT7v2_CD4SP-L-CD28-CS(G)-tTAEmma E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

For each construct, an aliquot of competent DH10β E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells (1–5 μL) and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 μL of room temperature SOC, the mixture was then recovered for 30–60 min at 37 °C with shaking (#Heatblock). The mixture was centrifuged at 16000 x g for 30 seconds to remove the supernatant. The pellet was resuspended in 50 μl of LB and spread onto a selection plate and incubated overnight at 37°C.

Results:

E. coli colony picking for 630_pcDNAT7v2_iCasp9-T2A-GFP, 640_pcDNAT7v2_EGFP, and 650_pcDNAT7v2_CD4SP-L-CD28-CS(G)-tTAEmma>

2025-09-11

Emma Schwarze

Goal

Colony picking of individual DH10β E. coli (Cat. No. C3019, New England Biolabs) bacterial colonies transformed with 630_pcDNAT7v2_iCasp9-T2A-GFP, 640_pcDNAT7v2_EGFP, and 650_pcDNAT7v2_CD4SP-L-CD28-CS(G)-tTAEmma for the replication and expression of the desired genetic material.

Protocol

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plates. Each colony was transferred to 5 mL of fresh culture medium (LB medium with 100 μg/mL carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm (CERTOMAT BS-1 Incubator Shaker, Sartorius) for further growth and amplification of the selected cells. The agar plates were sealed to prevent contamination and stored at 4 °C.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial cultures after overnight incubation at 37 °C.

Plasmid Miniprep (NEB) of 630_pcDNAT7v2_iCasp9-T2A-GFP, 640_pcDNAT7v2_EGFP, and 650_pcDNAT7v2_CD4SP-L-CD28-CS(G)-tTAEmma>

2025-09-12

Emma Schwarze

Goal:

Minipreps of 630_pcDNAT7v2_iCasp9-T2A-GFP, 640_pcDNAT7v2_EGFP, and 650_pcDNAT7v2_CD4SP-L-CD28-CS(G)-tTAEmma >

Protocol:

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight cultures were pelleted by centrifugation for 15 minutes at 4000 ×  g (Eppendorf, Centrifuge 5804R). For each sample, the supernatant was discarded. Pellet was resuspended in 200 μL of plasmid resuspension buffer. Cell lysis was achieved by the addition of 200 μL of plasmid lysis buffer, followed by 5–6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of plasmid neutralization buffer. The tubes were gently inverted and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transferred to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of plasmid wash buffer 1 and centrifuging for 1 minute, followed by 400 μL of plasmid wash buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL Eppendorf tubes. DNA was eluted by the addition of 30 μL of preheated TE buffer (55–65 °C) to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop (#Nano_model), and the DNA samples were stored at −20 °C.

Results:

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
630.1	668.13	1.93	2.35
630.2	610.70	1.94	2.37
630.3	576.41	1.90	2.36
640.1	340.37	1.87	2.54
640.2	536.95	1.90	2.41
640.3	580.26	1.92	2.34
650.1	600.62	1.93	2.31
650.2	654.29	1.94	2.30
650.3	628.99	1.93	2.29

PCR (Platinum™ SuperFi™ II) of 950_pcDNAT7v2_MxSig-BmTyr-DD-C, 960_pcDNAT7v2_QtSig-BmTyr-DD-C, 970_pcDNAT7v2_TmSig-BmTyr-DD-C

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments  for constructs 950_pcDNAT7v2_MxSig-BmTyr-DD-C, 960_pcDNAT7v2_QtSig-BmTyr-DD-C, 970_pcDNAT7v2_TmSig-BmTyr-DD-C with high fidelity and efficiency.

Construct	Template	Primer	Length	Time
950	940	951 & 952	7.3	4 min
960	940	961 & 962	7.3	4 min
970	940	971 & 972	7.3	4 min

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA	10 ng	X μL
Nuclease-free water	–	X μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	5–10 sec	25–35
Annealing	60°C	10 sec
Extension	72°C	15–30 seconds/kb
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

E. coli colony picking of 660_pET_21(+)_Twin-Strep, 910_pcDNAT7v2_BmTyr-TEVCS(M)-MxEnc-eUnaG-STII-NES, 920_pcDNAT7v2_BmTyr-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 930_pcDNAT7v2_BmTyr-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal

Colony picking of individual DH10β E. coli (Cat. No. C3019, New England Biolabs) bacterial colonies transformed with 660_pET_21(+)_Twin-Strep, 910_pcDNAT7v2_BmTyr-TEVCS(M)-MxEnc-eUnaG-STII-NES, 920_pcDNAT7v2_BmTyr-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 930_pcDNAT7v2_BmTyr-TEVCS(M)-TmEnc-eUnaG-STII-NES from selection plates containing carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plates. Each colony was transferred to 5 mL of fresh culture medium (LB medium with 100 μg/mL carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm (CERTOMAT BS-1 Incubator Shaker, Sartorius) for further growth and amplification of the selected cells. The agar plates were sealed to prevent contamination and stored at 4 °C.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial cultures after overnight incubation at 37 °C.

Agarose Gel Electrophoresis of digested 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, digested 610, @153, @154, 200_pcDNAT7v1, and 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples-  digested 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, digested 610, @153, @154, 200_pcDNAT7v1, and 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, based on their size.

Protocol:

Agarose gel electrophoresis was performed using an 1% agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5–10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right:

L - dig.600.2 - dig.600.3 - dig.610.1 - dig.610.3 - X - L - 153 - 200(280) - 154 - 200 (290) - 250 (270)

Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples 600.3, 153, 200(280), 154, 200(290), and 250(270).

Gibson Assembly of 630_pcDNAT7v2_iCasp9-T2A-GFP, 950_pcDNAT7v2_MxSig-BmTyr-DD-C, 960_pcDNAT7v2_QtSig-BmTyr-DD-C, 970_pcDNAT7v2_TmSig-BmTyr-DD-C, 720_pET_21(+)_NBmTyr(85)-P4-Twin-Strep, 730_pET_21(+)_P3-CBmTyr(85)-Twin-Strep, 750_pET_21(+)_P3-CBmTyr(157)-Twin-Strep, 760_pET_21(+)_NBmTyr(201)-P4-Twin-Strep, 770_pET_21(+)_P3-CBmTyr(201)-Twin-Strep

Goal:

Gibson assembly was performed to produce 630_pcDNAT7v2_iCasp9-T2A-GFP, 950_pcDNAT7v2_MxSig-BmTyr-DD-C, 960_pcDNAT7v2_QtSig-BmTyr-DD-C, 970_pcDNAT7v2_TmSig-BmTyr-DD-C, 720_pET_21(+)_NBmTyr(85)-P4-Twin-Strep, 730_pET_21(+)_P3-CBmTyr(85)-Twin-Strep, 750_pET_21(+)_P3-CBmTyr(157)-Twin-Strep, 760_pET_21(+)_NBmTyr(201)-P4-Twin-Strep, 770_pET_21(+)_P3-CBmTyr(201)-Twin-Strep

Protocol:

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Components	Volume	630	720	730	750	760	770	950	960	970
NEBuilder HiFi DNA Assembly Master Mix	10 µL
Vector	50 ng	1.5	0.5	0.6	0.6	0.8	5.3	0.7	1	0.6
Insert	150 ng	175: 1.5	x	x	x	x	x	x	x	x
Insert 2	150 ng	176: 0.6	x	x	x	x	x	x	x	x
Nuclease-free Water	... μL	6.4	9.5	9.4	9.4	9.2	4.7	9.3	9	9.4
Total	20 µL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of Gibson assembly was evaluated by downstream experiments.

Plasmid Miniprep (NEB) of 660_pET_21(+)_Twin-Strep, 910_pcDNAT7v2_BmTyr-TEVCS(M)-MxEnc-eUnaG-STII-NES, 920_pcDNAT7v2_BmTyr-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 930_pcDNAT7v2_BmTyr-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Minipreps of 660_pET_21(+)_Twin-Strep, 910_pcDNAT7v2_BmTyr-TEVCS(M)-MxEnc-eUnaG-STII-NES, 920_pcDNAT7v2_BmTyr-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 930_pcDNAT7v2_BmTyr-TEVCS(M)-TmEnc-eUnaG-STII-NES were performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol:

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight cultures were pelleted by centrifugation for 15 minutes at 4000 ×  g (Eppendorf, Centrifuge 5804R). For each sample, the supernatant was discarded. Pellet was resuspended in 200 μL of plasmid resuspension buffer. Cell lysis was achieved by the addition of 200 μL of plasmid lysis buffer, followed by 5–6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of plasmid neutralization buffer. The tubes were gently inverted and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transferred to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of plasmid wash buffer 1 and centrifuging for 1 minute, followed by 400 μL of plasmid wash buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL Eppendorf tubes. DNA was eluted by the addition of 30 μL of preheated TE buffer (55–65 °C) to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop (#Nano_model), and the DNA samples were stored at −20 °C.

Results:

The miniprep procedure yielded DNA of sufficient quantity within the acceptable range.

Sample	Concentration [ng/μL]
660.1	49.44
660.2	47.44
910.1	509.01
910.2	526.45
910.3	556.81
910.4	487.59
920.1	583.99
920.2	543.98
920.3	657.54
920.4	539.49
930.1	518.80
930.2	501.39
930.3	521.63
930.4	295.94

PCR Clean-up (NEB) of 950_pcDNAT7v2_MxSig-BmTyr-DD-C, 960_pcDNAT7v2_QtSig-BmTyr-DD-C, and 970_pcDNAT7v2_TmSig-BmTyr-DD-C

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity within the acceptable range.

Sample	Concentration [ng/μL]
950	198.82
960	126.16
970	198.84

PCR Clean-up (NEB) of 720_pET_21(+)_NBmTyr(85)-P4-Twin-Strep, 730_pET_21(+)_P3-CBmTyr(85)-Twin-Strep, 740_pET_21(+)_NBmTyr(157)-P4-Twin-Strep, 750_pET_21(+)_P3-CBmTyr(157)-Twin-Strep, 760_pET_21(+)_NBmTyr(201)-P4-Twin-Strep, and 770_pET_21(+)_P3-CBmTyr(201)-Twin-Strep

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity within the acceptable range.

Sample	Concentration [ng/μL]
720	191.99
730	171.94
740	8.27
750	175.00
760	193.31
770	155.37

PCR (Platinum™ SuperFi™ II) of 720_pET_21(+)_NBmTyr(85)-P4-Twin-Strep, 730_pET_21(+)_P3-CBmTyr(85)-Twin-Strep, 740_pET_21(+)_NBmTyr(157)-P4-Twin-Strep, 750_pET_21(+)_P3-CBmTyr(157)-Twin-Strep, 760_pET_21(+)_NBmTyr(201)-P4-Twin-Strep, 770_pET_21(+)_P3-CBmTyr(201)-Twin-Strep

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments for constructs 720_pET_21(+)_NBmTyr(85)-P4-Twin-Strep, 730_pET_21(+)_P3-CBmTyr(85)-Twin-Strep, 740_pET_21(+)_NBmTyr(157)-P4-Twin-Strep, 750_pET_21(+)_P3-CBmTyr(157)-Twin-Strep, 760_pET_21(+)_NBmTyr(201)-P4-Twin-Strep, 770_pET_21(+)_P3-CBmTyr(201)-Twin-Strep with high fidelity and efficiency.

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA	10 ng	X μL
Nuclease-free water	–	X μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	5–10 sec	25–35
Annealing	60°C	10 sec
Extension	72°C	15–30 seconds/kb
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

E. coli colony picking of more tyrosinases

Goal

Colony picking of individual DH10β E. coli (Cat. No. C3019, New England Biolabs) bacterial colonies transformed with our tyrosinase constructs from selection plates containing carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plates. Each colony was transferred to 5 mL of fresh culture medium (LB medium with 100 μg/mL carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm (CERTOMAT BS-1 Incubator Shaker, Sartorius) for further growth and amplification of the selected cells. The agar plates were sealed to prevent contamination and stored at 4 °C.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial cultures after overnight incubation at 37 °C.

Plasmid Miniprep (NEB) of tyrosinases

Goal:

Minipreps of Tyrosinases were performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol:

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight cultures were pelleted by centrifugation for 15 minutes at 4000 ×  g (Eppendorf, Centrifuge 5804R). For each sample, the supernatant was discarded. Pellet was resuspended in 200 μL of plasmid resuspension buffer. Cell lysis was achieved by the addition of 200 μL of plasmid lysis buffer, followed by 5–6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of plasmid neutralization buffer. The tubes were gently inverted and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transferred to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of plasmid wash buffer 1 and centrifuging for 1 minute, followed by 400 μL of plasmid wash buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL Eppendorf tubes. DNA was eluted by the addition of 30 μL of preheated TE buffer (55–65 °C) to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop (#Nano_model), and the DNA samples were stored at −20 °C.

Results:

The miniprep procedure yielded DNA of sufficient quantity within the acceptable range.

Sample	Concentration [ng/μL]
690	335.50
790.1	33.80
790.2	51.19
790.3	47.50
810.1	42.08
810.2	62.42
820.1	109.13
820.2	55.95
820.3	53.24
830.1	51.82
830.2	40.66
840.1	33.14
840.2	62.88
860.1	40.87
860.2	29.39
880.1	29.51
880.2	24.08
880.3	13.50
890.1	48.35
890.2	7.42
890.3	24.25

TXTL of 330_LET_CLS-XaCS-BmTyr - 580_pAAVS1_CAGp-HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K)_IRES-BSD Copy

Goal:

Cell-free transcription-translation (TXTL) reaction was performed to in vitro-express tyrosinase for enzyme activity testing of 330-580.

Protocol:

TXTL was performed using proprietary E.coli cell extract and Buffer B composition (Invitris GmbH). For this, a master mix containing Buffer B, T7 Polymerase and cell extract was prepared. The master mix composition was as follows:

Component	Volume
Buffer B	241.5 μL
Cell extract	172.5 μL
T7 Polymerase	5.75 μL

The mixture was then incubated on ice for 5-10 mins. Master mix was then distributed to PCR tubes, 12.3 μL each, and 2.7 μL DNA was added to the concentration of 20 nM.

Sample	Concentration of DNA [nM]	End concentration [nM]
330_LET_CLS-XaCS-BmTyr	271.06	20
340_LET_CLS-XaCS-BmTyr(E195S+A221V)	461.90	20
350_LET_CLS-XaCS-BmTyr(R209S+M215E+V218M)	328.39	20
360_LET_CLS-XaCS-BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C)	312.99	20
370_LET_CLS-XaCS-cpBmTyr(201)	275.90	20
380_LET_CLS-XaCS-NBmTyr(85)-P4	443.19	20
390_LET_CLS-XaCS-P3-CBmTyr(85)	356.22	20
380_LET_CLS-XaCS-NBmTyr(85)-P4/390_LET_CLS-XaCS-P3-CBmTyr(85)	—	20/20
400_LET_CLS-XaCS-NBmTyr(157)-P4	414.71	20
410_LET_CLS-XaCS-P3-CBmTyr(157)	358.60	20
400_LET_CLS-XaCS-NBmTyr(157)-P4/410_LET_CLS-XaCS-P3-CBmTyr(157)	—	20/20
420_LET_CLS-XaCS-NBmTyr(201)-P4	375.70	20
430_LET_CLS-XaCS-P3-CBmTyr(201)	449.11	20
420_LET_CLS-XaCS-NBmTyr(201)-P4/430_LET_CLS-XaCS-P3-CBmTyr(201)	—	20/20
440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466) (–TEV)	535.55	20
440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466) (+TEV, 10 U)	535.55	20
450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)	248.79	20
460_LET_CLS-XaCS-LsTyr	152.40	20
470_LET_CLS-XaCS-SkMelC1(33-124)	461.43	20
480_LET_CLS-XaCS-SkMelC2	276.16	20
470_LET_CLS-XaCS-SkMelC1(33-124)/480_LET_CLS-XaCS-SkMelC2	—	20/20
490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518)  (–TEV)	248.88	20
490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518) (+TEV, 10 U)	248.88	20
500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)	250.68	20
510_LET_CLS-XaCS-SavMelC1(27-118)	466.88	20
530_LET_CLS-XaCS-SavMelC2	303.59	20
510_LET_CLS-XaCS-SavMelC1(27-118)/530_LET_CLS-XaCS-SavMelC2	—	20/20
520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F)	455.23	20
540_LET_CLS-XaCS-SavMelC2(I42Y)	353.89	20
520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F)/540_LET_CLS-XaCS-SavMelC2(I42Y)	—	20/20

The samples was mixed carefully by stirring. The samples were incubated at 29°C for 8h in a thermocycler followed by a 4°C storage.

Results:

The efficiency of TXTL was evaluated by downstream experiments.

TXTL of 330_LET_CLS-XaCS-BmTyr

Goal:

Cell-free transcription-translation (TXTL) reaction was performed to troubleshoot in vitro-expression of tyrosinases.

Protocol:

TXTL was performed using proprietary E.coli cell extract (Invitris GmbH). For this, a master mix containing Buffer B (Invitris GmbH), T7 Polymerase and cell extract was prepared. The master mix composition was as follows:

Component	Volume
Buffer B	40.25 μL
Cell extract	28.75 μL
T7 Polymerase	0.96 μL

The mixture was then incubated on ice for 5-10 mins. Master mix was then distributed to PCR tubes, 12.3 μL each, and 2.7 μL DNA (330_LET_CLS-XaCS-BmTyr) was added to the concentration of 20 nM. The samples was mixed carefully by stirring. The samples were incubated at 29°C for 8h in a thermocycler followed by a 4°C storage.

Results:

The efficiency of TXTL was evaluated by downstream experiments.

Plasmid Maxiprep (QIAGEN) of 660_pET_21(+)_Twin-Strep

Goal:

A maxiprep of ... was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Protocol:

DNA plasmids were prepared using the QIAGEN ® Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). ... mL of overnight culture of E. coli was centrifuged at 4500 × g for 20 min at 4°C. The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer with thorough mixing. The mixture was centrifuged at 7000 × g (eppendorf 5430 R) for 135 min (if supernatant not clear, optional +30) min at 4 °C. The supernatant was applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuging at 7000 × g for 75 min (if pellet not strong enough, additional 30 min) at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol. Finally, the supernatant was aspirated using a vacuum pump without disturbing the pellet, and the pellet was air-dried for 5 - 10 min. The DNA was then redissolved in 200 μL of TE buffer, the concentration was determined using a NanoDrop, and the DNA was stored at −20 °C.

Results:

The maxiprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230

PCR (Platinum™ SuperFi™ II) for 980_pcDNAT7v2_MxEnc-eUnaG-STII-NES, 990_pcDNAT7v2_QtEnc-eUnaG-STII-NES, and 1000_pcDNAT7v2_TmEnc-eUnaG-STII-NES

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments with high fidelity and efficiency.

Composite part	template	primer	length	extension time	tube name
980	590	981 & 982	7.4	4 min	98
990	600	991 & 982	7.4	4 min	99
1000	610	1001 & 982	7.4	4 min	100

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA		0.5 μL
DMSO		1 µL
Nuclease-free water	–	6.5 μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	10 sec	25
Annealing	58°C	20 sec
Extension	72°C	30 seconds/kb
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

PCR (Platinum™ SuperFi™ II) for 940_pcDNAT7v2_TyrBm-DD-C, 980_pcDNAT7v2_MxEnc-eUnaG-STII-NES, 990_pcDNAT7v2_QtEnc-eUnaG-STII-NES, and 1000_pcDNAT7v2_TmEnc-eUnaG-STII-NES

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments with high fidelity and efficiency.

Composite part	template	primer	length	extension time	tube name
940	590	941 & 942	6.1	3 min 15 sec	94
980	590	981 & 982	7.4	4 min	98
990	600	991 & 982	7.4	4 min	99
1000	610	1001 & 982	7.4	4 min	100

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA		0.5 μL
DMSO		0.5 µL
Nuclease-free water	–	X μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	10 sec	25
Annealing	60°C	20 sec
Extension	72°C	30 seconds/kb
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

PCR (Platinum™ SuperFi™ II) of @172 and @173

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from @172 and @173 for constructs 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES with high fidelity and efficiency.

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)146 & 147	0.5 μM	2 μL
Template DNA	10 ng	1 μL
DMSO		0.5 µL
Nuclease-free water	–	6.5 μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98 °C	30 sec	1
Denaturation	98 °C	10 sec	25
Annealing	58 °C	20 sec
Extension	72 °C	1 min
Final extension	72 °C	5 min	1
Hold	4 °C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

PCR (Platinum™ SuperFi™ II) for 910_pcDNAT7v2_BmTyr-TEVCS(M)-MxEnc-eUnaG-STII-NES, 920_pcDNAT7v2_BmTyr-TEVCS(M)-QtEnc-eUnaG-STII-NES, 930_pcDNAT7v2_BmTyr-TEVCS(M)-TmEnc-eUnaG-STII-NES, 940_pcDNAT7v2_TyrBm-DD-C, 980_pcDNAT7v2_MxEnc-eUnaG-STII-NES, 990_pcDNAT7v2_QtEnc-eUnaG-STII-NES, and 1000_pcDNAT7v2_TmEnc-eUnaG-STII-NES

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments with high fidelity and efficiency.

Composite part	template	primer	length	extension time	tube name
910	620	913 & 914	0.9	40 sec	910i
	590	911 & 912	7.4	4 min	910bb
920	600	921 & 912	7.4	4 min	920
930	610	931 & 912	7.4	4 min	930
940	590	941 & 942	6.1	3 min 15 sec	940bb
	620	943 & 944	1.2	45 sec	940i
980	590	981 & 982	7.4	4 min	980
990	600	991 & 982	7.4	4 min	990
1000	610	1001 & 982	7.4	4 min	1000

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA		0.5 μL
DMSO		0.5 µL
Nuclease-free water	–	X μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	10 sec	25
Annealing	60°C	20 sec
Extension	72°C	30 seconds/kb
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

Splitting of HEK293T cell line into T25 flask - p13 & p30

Goal:

Splitting HEK293t cell line from "HEK VI, VII, VIII, IV" flask to the next passage number of p13 & "HEK V" flask to the next passage number of p30

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Culturing media was decanted from the 25 cm 2 culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos's modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 25 cm 2 flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO 2 and 90% relative humidity.

Results:

HEK293t cell line from "[Name of the flask]" flask was split to passage number __

Splitting of HEK293T cell line into T25 flask - p12 & p29

Goal:

Splitting HEK293t cell line from "HEK VI & VII" flask  to the next passage number of p12

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Culturing media was decanted from the 25 cm 2 culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos's modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 25 cm 2 flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO 2 and 90% relative humidity.

Results:

HEK293t cell line from "HEK VI & VII" flask  to the next passage number of p12

Splitting of HEK293T cell line into T25 flask - p28

Goal:

Splitting HEK293t cell line from "HEK I-V" flask  to the next passage number of p28

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Culturing media was decanted from the 25 cm 2 culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos's modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 25 cm 2 flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO 2 and 90% relative humidity.

Results:

HEK293t cell line from "[Name of the flask]" flask was split to passage number __

Seeding of Nunc MicroWell 96-Well plates

Goal:

Seeding Nunc™ MicroWell™ 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate (Thermo Scientific™) for further confocal analysis of 620_pBudCE4.1_MxEncA_STII-M transfected into HEK293T cells with \(1 \times 10^4\) HEK293T.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet. Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 37 °C for 5 minutes. Upon confirming detachment using a light phase microscope, cells were rescued using 3 ml pre-warmed complete Dulbecco's modified Eagle medium F12 (DMEM) and transferred to a 15 ml tube. Cell suspension was centrifuged at 25 °C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using a Pasteur pipette and the cell pellet was resuspended in 6 ml pre-warmed DMEM. Each well of a Nunc™ 96-well plate was inoculated with 100 µl of cell suspension at \(1 \times 10^4\) cells per well.  Cells were further grown at 37 °C with 5% CO₂ and 90% relative humidity.

Results:

Countess™ II automated cell counter results:

Chamber	Cell count (\(\times 10^6\))	Live cells (\(\times 10^6\))	Dead cells (\(\times 10^3\))
A	_x10⁶	_x10⁶	_x10³
B	_x10⁶	_x10⁶	_x10³

Plasmid Maxiprep (QIAGEN) of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

A maxiprep of ... was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Protocol:

DNA plasmids were prepared using the QIAGEN ® Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). ... mL of overnight culture of E. coli was centrifuged at 4500 × g for 20 min at 4°C. The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer with thorough mixing. The mixture was centrifuged at 7000 × g (eppendorf 5430 R) for 135 min (if supernatant not clear, optional +30) min at 4 °C. The supernatant was applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuging at 7000 × g for 75 min (if pellet not strong enough, additional 30 min) at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol. Finally, the supernatant was aspirated using a vacuum pump without disturbing the pellet, and the pellet was air-dried for 5 - 10 min. The DNA was then redissolved in 200 μL of TE buffer, the concentration was determined using a NanoDrop, and the DNA was stored at −20 °C.

Results:

The maxiprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
270	612	1.9	2.3
600	1232	1.9	2.3
610	966	1.9	2.3

PCR Clean-up (NEB) of digested 320_pcDNAT7v2

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
320_pcDNAT7v2	32.9	1.96	-13.44

PCR Clean-up (NEB) of the stupid tyrosinases

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
1	7.64	1.91	-0.38
1_new	39.80	1.87	-10.11
2	60.61	1.91	11.04
3	305.45	1.94	2.89
4	224.98	1.95	2.90
5	239.38	1.95	2.99
6	230.80	1.94	3.05
7	210.78	1.94	2.96
8	156.73	1.93	3.03
9	213.27	1.92	2.92
10	216.00	1.95	3.02
11	199.64	1.90	2.84
12	171.66	1.95	3.42
13	181.95	1.94	3.21
14	169.25	1.96	3.25
15	221.27	1.96	2.97
16	224.71	1.94	2.95
17	167.72	1.96	3.17
18	212.86	1.97	2.95
19	161.47	1.95	3.13
20	205.33	1.95	2.93
21	124.15	1.95	3.56
22	188.49	1.97	2.93
23	173.30	1.95	3.14
24	179.69	1.95	3.11

PCR Clean-up (NEB) of 560_pAAVS1_CAGp-IL7Ra-IRES-BSD and @154

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
560	57.60	1.96	12.66
154	185.85	1.94	3.05

PCR (Platinum™ SuperFi™ II) of 560_pAAVS1_CAGp-IL7Ra-IRES-BSD and @154

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from ... for constructs 570_pAAVS1_CAGp-HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)_IRES-BSD with high fidelity and efficiency.

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA	10 ng	X μL
Nuclease-free water	–	X μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	5–10 sec	25–35
Annealing	60°C	10 sec
Extension	72°C	15–30 seconds/kb
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

Agarose Gel Electrophoresis of these damn tyrosinases & Killswitch

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Protocol:

Agarose gel electrophoresis was performed using a ...% w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5–10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right:

First gel: L-NC-1-1_new-X-L-2-X-L-560-154

Second gel:

Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

Analytical Agarose Gel: Digest of 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES.1 - 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES.6

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Protocol:

Agarose gel electrophoresis was performed using a ...% w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5–10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right: Ladder - 610.1 - 610.2 - 610.3 - 610.4 - 610.5 - 610.6 -> only 610.4, 610.5 and 610.6 showed 2 band, they were running higher than expected though

Sanger sequencing: 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES.1, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES.2, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES.5, 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES.1, 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES.3, 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K).3, 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K).4, 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES.5

Goal:

To confirm the nucleotide sequences of …, Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a (partial) match between the experimental and expected sequences. In addition, the sequencing data showed (predominantly) a high quality score and a continuous read length of over 1 kb.

Sample	Barcode	Primer	Validation
600.1 (25.08.)	10344001	CMV
600.1 (25.08.)	10344002	WPRE
600.2 (25.08.)	10344003	CMV
600.2 (25.08.)	10344004	WPRE
600.5 (25.08.)	10344005	CMV
600.5 (25.08.)	10344006	WPRE
610.1 (25.08.)	10344007	CMV
610.1 (25.08.)	10344008	WPRE
610.3 (25.08.)	10344009	CMV
610.3 (25.08.)	10344010	WPRE
300.3 (25.08.)	10344011	CMV
300.3 (25.08.)	10344012	WPRE
300.4 (25.08.)	10344013	CMV
300.4 (25.08.)	10344014	WPRE
610.5 (28.08.)	10344015	CMV
610.5 (28.08.)	10344016	WPRE

Splitting of HEK293T cell line into T25 flask - p27

Goal:

Splitting HEK293t cell line from "HEK I - V" flask  to the next passage number of p27.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Culturing media was decanted from the 25 cm 2 culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos's modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 25 cm 2 flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO 2 and 90% relative humidity.

Results:

HEK293t cell line from "HEK I - V" flask was split to passage number p27.

Analytical digest of 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES.1 - 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES.6

Goal:

Analytical digest of the 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES.1 - 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES.6 with KpnI & HindIII enzymes.

Protocol:

Restriction digest was carried out using the KpnI & HindIII enzymes (…,  New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 1 hour.

Mastermix:

Component	Volume
Restriction enzymes (each)	3 μL
rCutSmart buffer	15 μL
Nuclease-free Water	to 50 μL

9µl of Mastermix was added to 1µl of digest.

Optionally, the reaction was stopped using heat inactivation (80 °C, 20 minutes). The products were then stored at –20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

Plasmid Miniprep (NEB): 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES.1 - 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES.6

Goal:

Minipreps of 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES.1 - 610.6  were performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol:

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight cultures were pelleted by centrifugation for 15 minutes at 4000 ×  g (Eppendorf, Centrifuge 5804R). For each sample, the supernatant was discarded. Pellet was resuspended in 200 μL of plasmid resuspension buffer. Cell lysis was achieved by the addition of 200 μL of plasmid lysis buffer, followed by 5–6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of plasmid neutralization buffer. The tubes were gently inverted and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transferred to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of plasmid wash buffer 1 and centrifuging for 1 minute, followed by 400 μL of plasmid wash buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL Eppendorf tubes. DNA was eluted by the addition of 30 μL of preheated TE buffer (55–65 °C) to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop (#Nano_model), and the DNA samples were stored at −20 °C.

Results:

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
610.1	227	1.93	2.16
610.2	240	1.95	2.60
610.3	135	1.85	3.00
610.4	209	1.93	2.61
610.5	275	2.09	2.69
610.6	210	1.93	2.62

Amplification of DNA using the phi29-XT RCA Kit for plasmid DNA directly from bacterial cells

Goal:

DNA of mid- or high-copy number plasmids 630_pcDNAT7v2_iCasp9-T2A-GFP was amplified using the phi29-XT RCA kit (NEB #E1603) for downstream applications.

Protocol:

For each sample, a sterile pipette tip was used to pick a bacterial colony from an agar plate containing X. The colony was resuspended in 100 µL nuclease-free water in a PCR tube.In case of a liquid culture, 1 µL of bacterial culture was transferred to 9 µL nuclease-free water.

The cells were lysed by incubation in a thermocycler (model) at 95 °C for 3 minutes, with a lid temperature of 100 °C. Afterwards, the samples were transferred to ice.

Reactions with an end volume of 20 µL were prepared as described in the table below. For each reaction, 4 µL of phi29-XT Reaction Buffer (5X) was added to achieve a final concentration of 1X, as well as 2 µL of Deoxynucleotide (dNTP) Solution Mix (10 mM) for a final concentration of 1 mM.  Furthermore, 2 µL of Exonuclease-Resistant Random Primers (500 µM) were added to achieve a final concentration of 50 µM, as well as 2 µL of phi29-XT DNA Polymerase (10X) for a final concentration of 1X. To get to a final concentration of X, X µL of the lysed cells was added. Nuclease-free water was used to bring the reaction to 18 µL.

Components	20 µL reaction	Final concentration
phi29-XT Reaction Buffer, 5X	4 µL	1X
Deoxynucleotide (dNTP) Solution Mix, 10 mM	2 µL	1 mM
Exonuclease-Resistant Random Primers, 500 µM	2 µL	50 µM
phi29-XT DNA Polymerase, 10X	2 µL	1X
Lysed cells	up to 10 µL	N/A
Nuclease-free Water	to 18 µL	N/A

Each reaction was mixed thoroughly by gentle pipetting or vortexing, followed by brief centrifuging.

Lastly, the reactions were incubated in a thermocycler (model) at 42 °C for 2 hours, with a lid temperature of ≥ 75°C, followed by 10 minutes at 65 °C to inactivate the DNA polymerase.

The RCA products can be kept at 4 °C overnight or at  -20 °C for long-term storage.

Results:

DNA Transfection of HEK293T with jetOPTIMUS: 620_pBudCE4.1_MxEncA_STII-M

Goal:

Introduction of 620_pBudCE4.1_MxEncA_STII-M into HEK293T cell line at cell partition p25

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Cells were seeded in 8 well microscopic slides from ibidi at density of 5*10 3 cells per well and grown overnight. Afterwards cells were treated with jetOPTIMUS reagent mixture, including plasmid of interest and fresh DMEM. Afterwards, cells were grown at  37°C with 5% CO 2 and 90% relative humidity.

Results:

[INSERT AND DESCRIBE PICTURES HERE PLEASE FROM YOUR POSITIVE CONTROL]

Seeding of Nunc MicroWell 96-Well plates

Goal:

Seeding Nunc™ MicroWell™ 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate (Thermo Scientific™) for further confocal analysis of [Plasmids/Treatments/Controls] transfected into HEK293T cells with \(1 \times 10^4\) HEK293T.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet. Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 37 °C for 5 minutes. Upon confirming detachment using a light phase microscope, cells were rescued using 3 ml pre-warmed complete Dulbecco's modified Eagle medium F12 (DMEM) and transferred to a 15 ml tube. Cell suspension was centrifuged at 25 °C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using a Pasteur pipette and the cell pellet was resuspended in 6 ml pre-warmed DMEM. Each well of a Nunc™ 96-well plate was inoculated with 100 µl of cell suspension at \(1 \times 10^4\) cells per well.  Cells were further grown at 37 °C with 5% CO₂ and 90% relative humidity.

Results:

Countess™ II automated cell counter results:

Chamber	Cell count (\(\times 10^6\))	Live cells (\(\times 10^6\))	Dead cells (\(\times 10^3\))
A	_x10⁶	_x10⁶	_x10³
B	_x10⁶	_x10⁶	_x10³

Agarose Gel Electrophoresis with 320_pcDNAT7v2, @175, and @176

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Protocol:

Agarose gel electrophoresis was performed using a ...% w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5–10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right:

L-320-175-176

Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

Plasmid Miniprep (NEB) of300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K), 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Minipreps of 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K), 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES were performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol:

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight cultures were pelleted by centrifugation for 15 minutes at 4000 ×  g (Eppendorf, Centrifuge 5804R). For each sample, the supernatant was discarded. Pellet was resuspended in 200 μL of plasmid resuspension buffer. Cell lysis was achieved by the addition of 200 μL of plasmid lysis buffer, followed by 5–6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of plasmid neutralization buffer. The tubes were gently inverted and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transferred to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of plasmid wash buffer 1 and centrifuging for 1 minute, followed by 400 μL of plasmid wash buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL Eppendorf tubes. DNA was eluted by the addition of 30 μL of preheated TE buffer (55–65 °C) to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop (#Nano_model), and the DNA samples were stored at −20 °C.

Results:

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
300.1	378.74	2.00	2.26
300.2	399.86	1.92	2.30
300.3
300.4
300.5	468.73	1.89	2.29
600.1	327.00	1.92	2.44
600.2	307.01	1.92	2.43
600.3	249.83	1.95	2.45
600.4	177.81	1.89	2.60
600.5	364.30	1.91	2.41
610.1	243.07	1.92	2.40
610.2	266.25	1.92	2.46
610.3	306.48	1.94	2.37
610.4	109.92	1.90	2.56
610.5	176.02	1.91	2.45

PCR Clean-up (NEB) of 320_pcDNAT7v2, @175, and @176

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
320	116.29	1.94	3.92
175	103.61	1.93	3.46
176	195.29	1.94	2.93

gBlocks resuspension of @175 and @176

Goal:

The gBlocks @175 and @176 from ... were resuspended in TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) to a final concentration of 10 ng/μL.

Protocol:

Lyophilized gBlocks were briefly centrifuged to collect the pellet at the bottom of the tube. The DNA was resuspended by vortexing in nuclease-free IDTE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) to reach the desired concentration of 10 ng/μL.

Results:

The resuspended gBlocks were stored at −20 °C and used for subsequent downstream experiments.

PCR (Platinum™ SuperFi™ II) of 320_pcDNAT7v2, @175, and @176

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 320_pcDNAT7v2, @175, and @176 for constructs 630_pcDNAT7v2_iCasp9-T2A-GFP with high fidelity and efficiency.

Subpart	Primer	Length	Extension time
320_pcDNAT7v2	636 & 635	6 kb	3 min 15 sec
@175	633 & 634	780 bp	40 sec
@176	631 & 632	1.2 kb	50 sec

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA	10 ng	X μL
DMSO		0.5 µL
Nuclease-free water	–	X μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	5–10 sec	25
Annealing	60°C	20 sec
Extension	72°C	30 seconds/kb
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

Gibson Assembly of 630_pcDNAT7v2_iCasp9-T2A-GFP

Goal:

Gibson assembly was performed to assemble the 320_pcDNAT7v2 vector and @175 and @176 inserts to produce 630_pcDNAT7v2_iCasp9-T2A-GFP.

Protocol:

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Components	Volume
NEBuilder HiFi DNA Assembly Master Mix	10 µL
Vector	… ng
Insert	… ng
Nuclease-free Water	... μL
Total	20 µL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of Gibson assembly was evaluated by downstream experiments.

Restriction digest: @172 & @173 with KpnI and HindIII

Goal:

Restriction digest of the cleaned-up PCRs woth @172 and @173 with KndI & HindIII enzyme(s), for downstream cloning of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES & 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES.

Protocol:

Restriction digest was carried out using the … enzyme(s) (…,  New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 1 hour.

Component	Volume
HindIII	0.25 µL
KndI	0.25 µL
rCutSmart buffer	1 µL
DNA	5 µL
Nuclease-free Water	to 10 µL

Optionally, the reaction was stopped using heat inactivation (80 °C, 20 minutes). The products were then stored at –20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

E. coli colony picking of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal

Colony picking of individual DH10β E. coli (Cat. No. C3019, New England Biolabs) bacterial colonies transformed with 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES from selection plates containing carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plates. Each colony was transferred to 5 mL of fresh culture medium (LB medium with 100 μg/mL carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm (CERTOMAT BS-1 Incubator Shaker, Sartorius) for further growth and amplification of the selected cells. The agar plates were sealed to prevent contamination and stored at 4 °C.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial cultures after overnight incubation at 37 °C.

E. coli transformation with 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) and 560_pAAVS1_CAGp-IL7Ra-IRES-BSD

Goal:

Transformation was performed to introduce plasmid DNA construct 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) into DH10β E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

For each construct, an aliquot of competent DH10β E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells (1–5 μL) and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 μL of room temperature SOC, the mixture was then recovered for 30–60 min at 37 °C with shaking (#Heatblock). For 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), the mixture was centrifuged at 16000 x g for 30 seconds to remove the supernatant. The pellet was resuspended in 50 μl of LB and spread onto a selection plate and incubated overnight at 37°C. For 560_pAAVS1_CAGp-IL7Ra-IRES-BSD, 10 µL of the recovered culture were added to 5 mL of LB medium containing Carb and incubated overnight.

Results:

DNA Ligation of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Ligation of the ...  with ...  to assemble ... .

Protocol:

Ligation was carried out using the T4 DNA Ligase (M0202, New England Biolabs) according to the manufacturers protocol. DNA fragments of interest, including ... ng of vector with 3-fold molar excess of insert(s), were combined with 2 μL of T4 DNA Ligase Buffer (10x), and 1 μL of nuclease-free water. Molar amount of each DNA fragment was calculated using the NEBioCalculator tool. The sample was mixed by pipetting, and incubated at room temperature for 10 minutes. The reaction was then stopped using heat inactivation at 65°C, 10 minutes.

Component	Volume
T4 DNA Ligase Buffer (10x)	2 μL
Vector DNA	50 ng (... pmol)
Insert DNA	70.83 ng (... pmol)
Nuclease-free Water	To 20 μL
T4 DNA Ligase	1 μL

The reaction was then stored at -20°C or immediately used for transformation (1-5 μL of ligation product).

Results:

The efficiency of restriction digest was evaluated by downstream experiments.

Plasmid Maxiprep (QIAGEN) 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 620_pBudCE4.1_MxEncA_STII-M

Goal:

A maxiprep of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 620_pBudCE4.1_MxEncA_STII-M was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Protocol:

DNA plasmids were prepared using the QIAGEN ® Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). 200 mL of overnight culture of E. coli was centrifuged at 4500 × g for 20 min at 4°C. The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer with thorough mixing. The mixture was centrifuged at 7000 × g (eppendorf 5430 R) for 135 min (if supernatant not clear, optional +30) min at 4 °C. The supernatant was applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuging at 7000 × g for 75 min (if pellet not strong enough, additional 30 min) at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol. Finally, the supernatant was aspirated using a vacuum pump without disturbing the pellet, and the pellet was air-dried for 5 - 10 min. The DNA was then redissolved in 200 μL of TE buffer, the concentration was determined using a NanoDrop, and the DNA was stored at −20 °C.

Results:

The maxiprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
270	621.16	1.87	2.11
590	2551.27	1.93	2.20
620	1141.77	1.91	2.18

Preparation of LB broth

Goal

Preparation of 2 L stock of Luria-Bertani broth.

Protocol

Luria-Bertani broth was prepared using LB-Medium (Cat. No. X968.3, Carl ROTH). The final composition of the broth was as follows:

Component	Quantity
LB Medium	25g/L
Ultrapure water	to 1.5 L

The broth was sterilized by autoclaving at 121 °C for 20 minutes.

Results

Following autoclaving, no precipitation or contamination was observed. Sterile LB broth was obtained and stored at 4 °C.

Preparation of LB broth

Goal

Preparation of 3 L stock of Luria-Bertani broth.

Protocol

Luria-Bertani broth was prepared using LB-Medium (Cat. No. X968.3, Carl ROTH). The final composition of the broth was as follows:

Component	Quantity
LB Medium	25g/L
Ultrapure water	to 3 L

The broth was sterilized by autoclaving at 121 °C for 15 minutes.

Results

Following autoclaving, no precipitation or contamination was observed. Sterile LB broth was obtained and stored at 4 °C.

Splitting of HEK293T cell line into T25 flask - p26

Goal:

Splitting HEK293t cell line from ""HEK I-V" flask  to the next passage number of p26.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Culturing media was decanted from the 25 cm 2 culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 2 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 4 ml pre-warmed complete Dulbeccos's modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 25 cm 2 flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO 2 and 90% relative humidity.

Results:

HEK293t cell line from ""HEK I-V" flask was split to passage number p26.

Splitting of HEK293T cell line into T25 flask - p25

Goal:

Splitting HEK293t cell line from "HEK I-V" flask  to the next passage number of p25.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Culturing media was decanted from the 25 cm 2 culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 1 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 5 ml pre-warmed complete Dulbeccos's modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 25 cm 2 flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO 2 and 90% relative humidity.

Results:

HEK293t cell line from "HEK I-V" flask was split to passage number p25.

E. coli colony picking of 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K), 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal

Colony picking of individual DH10β E. coli (Cat. No. C3019, New England Biolabs) bacterial colonies transformed with 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K), 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES from selection plates containing carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plates. Each colony was transferred to 5 mL of fresh culture medium (LB medium with 100 μg/mL carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm (CERTOMAT BS-1 Incubator Shaker, Sartorius) for further growth and amplification of the selected cells. The agar plates were sealed to prevent contamination and stored at 4 °C.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial cultures after overnight incubation at 37 °C.

DNA Ligation of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Ligation of the ...  with ...  to assemble ... .

Protocol:

Ligation was carried out using the T4 DNA Ligase (M0202, New England Biolabs) according to the manufacturers protocol. DNA fragments of interest, including ... ng of vector with 3-fold molar excess of insert(s), were combined with 2 μL of T4 DNA Ligase Buffer (10x), and 1 μL of nuclease-free water. Molar amount of each DNA fragment was calculated using the NEBioCalculator tool. The sample was mixed by pipetting, and incubated at room temperature for 10 minutes. The reaction was then stopped using heat inactivation at 65°C, 10 minutes.

Component	Volume
T4 DNA Ligase Buffer (10x)	2 μL
Vector DNA	... ng (... pmol)
Insert DNA	... ng (... pmol)
Nuclease-free Water	To 20 μL
T4 DNA Ligase	1 μL

The reaction was then stored at -20°C or immediately used for transformation (1-5 μL of ligation product).

Results:

The efficiency of restriction digest was evaluated by downstream experiments.

Plasmid Miniprep (NEB) of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Minipreps of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES were performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol:

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight cultures were pelleted by centrifugation for 15 minutes at 4000 ×  g (Eppendorf, Centrifuge 5804R). For each sample, the supernatant was discarded. Pellet was resuspended in 200 μL of plasmid resuspension buffer. Cell lysis was achieved by the addition of 200 μL of plasmid lysis buffer, followed by 5–6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of plasmid neutralization buffer. The tubes were gently inverted and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transferred to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of plasmid wash buffer 1 and centrifuging for 1 minute, followed by 400 μL of plasmid wash buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL Eppendorf tubes. DNA was eluted by the addition of 30 μL of preheated TE buffer (55–65 °C) to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop (#Nano_model), and the DNA samples were stored at −20 °C.

Results:

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
600.1	462.12	1.90	2.44
600.2	589.30	1.92	2.39
600.3	493.54	1.92	2.42
600.4	496.88	1.90	1.86
610.1	419.42	1.91	2.50
610.2	543.06	1.91	2.28
610.3	572.73	1.92	2.38
610.4	590.81	1.92	2.29

PCR (Platinum™ SuperFi™ II) of 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP for constructs 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP with high fidelity and efficiency.

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each) 271 & 272	0.5 μM	2 μL
Template DNA (250.4 Maxi)	10 ng	0.4 μL
DMSO	2.5 %	0.5 µL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	10 sec	27
Annealing	58°C	20 sec
Extension	72°C	4 min  30 sec
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

DpnI digest of 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP to generate 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP

Goal:

Restriction digest of the amplified 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP with DpnI enzymes, for downstream cloning of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP.

Protocol:

Restriction digest was carried out using the … enzyme(s) (…,  New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 1 hour.

Component	Volume
Restriction enzyme	10 U (1 μL)
rCutSmart buffer	5 μL
DNA	1 μg (7µL)
Nuclease-free Water	to 37 μL

Optionally, the reaction was stopped using heat inactivation (80 °C, 10 minutes). The products were then stored at –20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

E. coli transformation of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 560_pAAVS1_CAGp-IL7Ra-IRES-BSD, 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES, 620_pBudCE4.1_MxEncA_STII-M

Goal:

Transformation was performed to introduce plasmid DNA constructs 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 560_pAAVS1_CAGp-IL7Ra-IRES-BSD, 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES, and 620_pBudCE4.1_MxEncA_STII-M into DH10β E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

For each construct, an aliquot of competent DH10β E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells (1–5 μL) and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 μL of room temperature SOC, the mixture was then recovered for 30–60 min at 37 °C with shaking (#Heatblock). The mixture was centrifuged at 16000 x g for 30 seconds to remove the supernatant. The pellet was resuspended in 50 μl of LB and spread onto a selection plate for 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES and incubated overnight at 37°C. For 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 560_pAAVS1_CAGp-IL7Ra-IRES-BSD, 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, and 620_pBudCE4.1_MxEncA_STII-M, 150 µL of the cell mixture was added into 200 mL of LB medium containing Carb (or Zeo for 620_pBudCE4.1_MxEncA_STII-M) and incubated in the shaker overnight at 37 °C.

Results:

PCR Clean-up (NEB) of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP after DpnI digest

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
270	22.4	1.34	-2.44

gBlocks resuspension of @171, @172,@173

Goal:

The gBlocks @171, @172, @173 from Twist were resuspended in IDTE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) to a final concentration of 10 ng/μL.

Protocol:

Lyophilized gBlocks were briefly centrifuged to collect the pellet at the bottom of the tube. The DNA was resuspended by vortexing in nuclease-free IDTE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) to reach the desired concentration o10 ng/μL.

Results:

The resuspended gBlocks were stored at −20 °C and used for subsequent downstream experiments.

Preparation of LB broth

Goal

Preparation of 1.5 L stock of Luria-Bertani broth.

Protocol

Luria-Bertani broth was prepared using LB-Medium (Cat. No. X968.3, Carl ROTH). The final composition of the broth was as follows:

Component	Quantity
LB Medium	25g/L
Ultrapure water	to 1.5 L

The broth was sterilized by autoclaving at 121 °C for 15 minutes.

Results

Following autoclaving, no precipitation or contamination was observed. Sterile LB broth was obtained and stored at 4 °C.

Preparation of LB agar

Goal

Preparation of 0.5 L stock of Luria-Bertani agar.

Protocol

Luria-Bertani agar was prepared using LB-Agar (Cat. No. 6675.3, Carl ROTH). The final composition of the solution was as follows:

Component	Quantity
LB-Agar	40g/L
Ultrapure water	to 0.5 L

The solution was sterilized by autoclaving at 121 °C for 15 minutes.

Results

Following autoclaving, no contamination was observed. Sterile LB agar was obtained and stored at 4 °C.

Analytical digest of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Restriction digest of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES with KpnI and HindIII enzymes.

Protocol:

Restriction digest was carried out using the … enzyme(s) (…,  New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 100 ng and combined with 0.5 μL of rCutSmart (B7204, New England Biolabs) and 0.1 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 30 minutes.

The mastermix for the digest of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES was pipetted as follows:

Component	Volume
KpnI	0.9 µL
HindIII	0.9 μL
rCutSmart buffer	4.5 μL
Nuclease-free Water	74.7 μL

The products were then stored at –20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

Restriction digest of @171, @172, @173 with KpnI-HF & HindIII-HF

Goal:

Restriction digest of @171, @172, @173 with HindIII-HF and KpnI-HF enzyme(s), for downstream cloning of 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES.

Protocol:

Restriction digest was carried out using the … enzyme(s) (…,  New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 1 hour.

Component	Volume	171	172	173
Restriction enzyme	10 U (1 μL)	1	1	1
rCutSmart buffer	5 μL	5	5	5
DNA	1 μg	6.7	5.6	4
Nuclease-free Water	to 50 μL	37.3	38.4	40

Optionally, the reaction was stopped using heat inactivation (80 °C, 20 minutes). The products were then stored at –20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

PCR Clean-up (NEB) of @153, @154, @155, @171, @172, @173

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
171	149.15	1.94	3.13
172	179.02	1.93	2.96
153	184.82	1.92	2.98
154	103.91	1.92	3.77
155	152.23	1.91	3.07

E. coli transformation with 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4, 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Transformation was performed to introduce plasmid DNA constructs 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4, 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES into DH10β E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

For each construct, an aliquot of competent DH10β E. coli cells stored at −80 °C was thawed on ice. 3 µL of the desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells (1–5 μL) and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 μL of room temperature SOC, the mixture was then recovered for 30–60 min at 37 °C with shaking (#Heatblock). The mixture was centrifuged at 16000 x g for 30 seconds to remove the supernatant. The pellet was resuspended in 50 μl of LB and spread onto a selection plate and incubated overnight at 37°C.

Results:

DNA Ligation for constructs 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Ligation of the 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K)  with @171, @172, and @173 to assemble 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES.

Protocol:

Ligation was carried out using the T4 DNA Ligase (M0202, New England Biolabs) according to the manufacturers protocol. DNA fragments of interest, including ... ng of vector with 3-fold molar excess of insert(s), were combined with 2 μL of T4 DNA Ligase Buffer (10x), and 1 μL of nuclease-free water. Molar amount of each DNA fragment was calculated using the NEBioCalculator tool. The sample was mixed by pipetting, and incubated at room temperature for 10 minutes. The reaction was then stopped using heat inactivation at 65°C, 10 minutes.

Component	Volume	590	600	610
T4 DNA Ligase Buffer (10x)	2 μL
Vector DNA	50 ng (... pmol)	0.6 µL	0.6 µL	0.6 µL
Insert DNA	42.5 ng (... pmol)	1.1 µL	1.1 µL	1 µL
Nuclease-free Water	To 20 μL
T4 DNA Ligase	1 μL

The reaction was then stored at -20°C or immediately used for transformation (1-5 μL of ligation product).

Results:

The efficiency of restriction digest was evaluated by downstream experiments.

Plasmid Miniprep (NEB) of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Minipreps of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES were performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol:

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight cultures were pelleted by centrifugation for 15 minutes at 4000 ×  g (Eppendorf, Centrifuge 5804R). For each sample, the supernatant was discarded. Pellet was resuspended in 200 μL of plasmid resuspension buffer. Cell lysis was achieved by the addition of 200 μL of plasmid lysis buffer, followed by 5–6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of plasmid neutralization buffer. The tubes were gently inverted and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transferred to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of plasmid wash buffer 1 and centrifuging for 1 minute, followed by 400 μL of plasmid wash buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL Eppendorf tubes. DNA was eluted by the addition of 30 μL of preheated TE buffer (55–65 °C) to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop (#Nano_model), and the DNA samples were stored at −20 °C.

Results:

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
600.4	419.45	1.88	2.47
600.5	513.53	1.88	2.46
600.6	360.83	1.89	2.46
610.4	438.65	1.90	2.42
610.5	235.59	1.87	2.68
610.6	481.48	1.89	2.42

PCR Clean-up (NEB) of @153, @154, 200_pcDNAT7v1, and 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
153	195.35	1.94	3.07
200 for 280	185.94	1.95	3.19
154	174.48	1.96	3.17
200 for 290	94.54	1.83	3.10
250 for 270	192.79	1.94	2.84

Analytical digest of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Restriction digest of 600.2, 600.3, 610.1 and 610.3 with KpnI-HF and HindIII-HF enzyme(s).

Protocol:

Restriction digest was carried out using the KpnI-HF and HindIII-HF enzyme(s) (…,  New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 1 hour.

Master mix for 4 reactions was pipetted as followed:

Component	Volume
KpnI	1 μL
HindIII	1 µL
rCutSmart buffer	5 μL
Nuclease-free Water	to 40 μL

8 µL of Mastermix was added to 2 µL of sample. The reaction was done at 37 °C for 30 minutes, and stopped using heat inactivation (80 °C, 20 minutes). The products were then stored at –20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

Sanger sequencing of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES

Goal:

To confirm the nucleotide sequences of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a (partial) match between the experimental and expected sequences. In addition, the sequencing data showed (predominantly) a high quality score and a continuous read length of over 1 kb.

Sample	Barcode	Primer	Validation
600.3	06817613	WPRE
600.3	06817614	CMV

Gibson Assembly of 280_pcDNAT7v1_RS20-cpEFGP-CaM and 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal:

Gibson assembly was performed to assemble the 200_pcDNAT7v1 vector and @153 and @154 inserts to produce 280_pcDNAT7v1_RS20-cpEFGP-CaM and 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K).

Protocol:

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Components	280	290
NEBuilder HiFi DNA Assembly Master Mix	10 µL	10 µL
Vector	50 ng (0.85 µL)	50 ng (1.50 µL)
Insert	32.5 ng (0.55 µL)	37.5 ng (0.65 µL)
Nuclease-free Water	8.60 μL	7.85 µL
Total	20 µL	20 µL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of Gibson assembly was evaluated by downstream experiments.

KLD of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP

Goal:

KLD reaction was performed to assemble construct 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP from the PCR product 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, respectively.

Protocol:

KLD reaction was performed using the KLD Enzyme Mix (M0554, New England Biolabs), consisting of a kinase, a ligase, and a DpnI restriction enzyme, according to the manufacturer's instructions. For each reaction, KLD reaction buffer, KLD enzyme mix, nuclease-free water, and the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 μL
KLD Reaction Buffer (2x)	5 μL
KLD Enzyme Mix (10x)	1 μL
Nuclease-free water	3 μL
Total	10 μL

The sample was incubated at room temperature (25 °C) for 5–10 minutes. Optionally, to increase the efficiency of the DpnI digestion, the mixture was additionally incubated at 37 °C for 30–60 minutes. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of KLD reaction was evaluated by downstream experiments.

Plasmid Miniprep (NEB) of 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal:

Minipreps of 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) were performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol:

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight cultures were pelleted by centrifugation for 15 minutes at 4000 ×  g (Eppendorf, Centrifuge 5804R). For each sample, the supernatant was discarded. Pellet was resuspended in 200 μL of plasmid resuspension buffer. Cell lysis was achieved by the addition of 200 μL of plasmid lysis buffer, followed by 5–6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of plasmid neutralization buffer. The tubes were gently inverted and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transferred to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of plasmid wash buffer 1 and centrifuging for 1 minute, followed by 400 μL of plasmid wash buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL Eppendorf tubes. DNA was eluted by the addition of 30 μL of preheated TE buffer (55–65 °C) to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop (#Nano_model), and the DNA samples were stored at −20 °C.

Results:

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
290.1
290.2
290.3
290.4

Agarose Gel Electrophoresis of digested 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Protocol:

Agarose gel electrophoresis was performed using a ...% w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5–10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right: …. Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

Analytical digest of 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal:

Analytical digest of 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) with MIuI enzyme.

Protocol:

Restriction digest was carried out using the MIuI-HF enzyme (…,  New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 200 ng and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 0.2 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 15 minutes.

The mastermix for the digest of 290.1, 290.2, 290.3, and 290.4 was pipetted as followed.

Component	Volume
Restriction enzyme	10 U (1 μL)
rCutSmart buffer	5 μL
Nuclease-free Water	19 μL

5 µL mastermix was added to 5 µL of DNA.

The reaction was stopped using heat inactivation (80 °C, 15 minutes). The products were then stored at –20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

Sanger sequencing of 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal:

To confirm the nucleotide sequences of 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a (partial) match between the experimental and expected sequences. In addition, the sequencing data showed (predominantly) a high quality score and a continuous read length of over 1 kb.

Sample	Barcode	Primer	Validation
290.1	06817619	CMV	mutations
290.1	06817620	WPRE	mutations
290.3	06817621	CMV	no contigs
290.3	06817622	WPRE	no contigs

PCR Clean-up (NEB) of amplified 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP to generate 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
270	129.8	1.90	3.63

Splitting of HEK293T cell line into T25 flask - p24

Goal:

Splitting HEK293t cell line from "HEK I-V" flask  to the next passage number of p24.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Culturing media was decanted from the 25 cm 2 culturing flask. Cells were washed using 3 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 2 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 4ml pre-warmed complete Dulbeccos's modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 25 cm 2 flask was inoculated with the 1ml cell suspension to 5 ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO 2 and 90% relative humidity.

Results:

HEK293t cell line from "HEK I-V" flask was split to passage number p24. Cell line IV showed abnormal cell aggregations and was split into a fresh 25cm flask and kept seperate from the others.

Splitting of HEK293T cell line into T25 flask - p23

Goal:

Splitting HEK293t cell line from "HEK I-IV" flask  to the next passage number of p23.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Culturing media was decanted from the 25 cm 2 culturing flask. Cells were washed using 3 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 2 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos's modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 25 cm 2 flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO 2 and 90% relative humidity.Add 50 µL of PenStrep to HEK IV and V DMEM medium in 25cm flask.

Results:

HEK293t cell line from "HEK I-IV" flask was split to passage number p23. Culture II showed abnormal cell agglomeration and was transferred into a fresh 25cm flask.

Analytical digest of 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal:

Restriction digest of 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) with MIuI-HF enzyme(s).

Protocol:

Restriction digest was carried out using the … enzyme(s) (…,  New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 30 minutes.

Component	Volume
Restriction enzyme	10 U (1 μL)
rCutSmart buffer	5 μL
DNA	1 μg
Nuclease-free Water	to 50 μL

Optionally, the reaction was stopped using heat inactivation (80 °C, 20 minutes). The products were then stored at –20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

Agarose Gel Electrophoresis

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Protocol:

Agarose gel electrophoresis was performed using a ...% w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 2 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5–10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right:

L - 320.1 - 320.2 - L - 600.10 - 600.11 - 600.12 - 610.10 - 610.11 - 610.12

Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

Analytical digest of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Restriction digest of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES with KpnI and HindIII enzymes.

Protocol:

Restriction digest was carried out using the … enzyme(s) (…,  New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 100 ng and combined with 0.5 μL of rCutSmart (B7204, New England Biolabs) and 0.1 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 45 minutes.

The mastermix was pipetted as follows:

Component	Volume
KpnI	0.7 μL
HindIII	0.7 µL
rCutSmart buffer	3.5 μL
Nuclease-free Water	58.1 μL

9 µL of mastermix were added to 1 µL of DNA (concentration 100 ng/µL). The products were immediately loaded onto an agarose gel for validation.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

Plasmid Miniprep (NEB) of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Minipreps of 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES were performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol:

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight cultures were pelleted by centrifugation for 15 minutes at 4000 ×  g (Eppendorf, Centrifuge 5804R). For each sample, the supernatant was discarded. Pellet was resuspended in 200 μL of plasmid resuspension buffer. Cell lysis was achieved by the addition of 200 μL of plasmid lysis buffer, followed by 5–6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of plasmid neutralization buffer. The tubes were gently inverted and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transferred to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of plasmid wash buffer 1 and centrifuging for 1 minute, followed by 400 μL of plasmid wash buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL Eppendorf tubes. DNA was eluted by the addition of 30 μL of preheated TE buffer (55–65 °C) to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop (#Nano_model), and the DNA samples were stored at −20 °C.

Results:

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
600.10	425.15	1.91	2.44
600.11	517.53	1.92	2.46
600.12	542.84	1.92	2.44
610.10	585.11	1.91	2.41
610.11	542.37	1.90	2.43
610.12	451.72	1.91	2.50

E. coli transformation with 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Transformation was performed to introduce plasmid DNA constructs … into DH10β E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

For each construct, an aliquot of competent DH10β E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells (1–5 μL) and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 μL of room temperature SOC, the mixture was then recovered for 30–60 min at 37 °C with shaking (#Heatblock). The mixture was centrifuged at 16000 x g for 30 seconds to remove the supernatant. The pellet was resuspended in 50 μl of LB and spread onto a selection plate and incubated overnight at 37°C.

Results:

PCR Clean-up (NEB) of 200_pcDNAT7v1 and @154

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
200	88.37	1.87	7.19
154	143.63	1.90	3.68

Agarose Gel Electrophoresis of 200_pcDNAT7v1, @154, digested 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Protocol:

Agarose gel electrophoresis was performed using a ...% w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 2 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 6 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 110 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right: L - 200 - 154 - L - 600.7 - 600.8 - 600.9 - 610.7 - 610.8 - 610.9

Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples for 200_pcDNAT7v1 and @154.

Gibson Assembly of 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal:

Gibson assembly was performed to assemble the 200_pcDNAT7v1 vector and @154 insert to produce 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K).

Protocol:

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 88.37 ng of vector with 3-fold molar excess of insert, were combined with 10 μL of master mix.

Components	Volume
NEBuilder HiFi DNA Assembly Master Mix	10 µL
Vector	1 µL (88.37 ng)
Insert	1.85 µL (265.71 ng)
Nuclease-free Water	7.15 μL
Total	20 µL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were immediately used for subsequent transformation.

Results:

The efficiency of Gibson assembly was evaluated by downstream experiments.

Plasmid Miniprep (NEB) of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Minipreps of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES were performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol:

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight cultures were pelleted by centrifugation for 15 minutes at 4000 ×  g (Eppendorf, Centrifuge 5804R). For each sample, the supernatant was discarded. Pellet was resuspended in 200 μL of plasmid resuspension buffer. Cell lysis was achieved by the addition of 200 μL of plasmid lysis buffer, followed by 5–6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of plasmid neutralization buffer. The tubes were gently inverted and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transferred to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of plasmid wash buffer 1 and centrifuging for 1 minute, followed by 400 μL of plasmid wash buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL Eppendorf tubes. DNA was eluted by the addition of 30 μL of preheated TE buffer (55–65 °C) to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop (#Nano_model), and the DNA samples were stored at −20 °C.

Results:

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
270.7	683.15	1.92	2.35
270.8	748.50	1.92	2.38
270.9	603.63	1.91	2.35
600.7	577.95	1.90	2.40
600.8	655.42	1.90	2.35
600.9	356.91	1.68	1.69
610.7	594.43	1.91	2.41
610.8	580.84	1.89	2.33
610.9	753.62	1.90	2.32

E. coli transformation of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES, and 560_pAAVS1_CAGp-IL7Ra-IRES-BSD

Goal:

Transformation was performed to introduce plasmid DNA constructs … into DH10β E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

For each construct, an aliquot of competent DH10β E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells (1–5 μL) and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 μL of room temperature SOC, the mixture was then recovered for 30–60 min at 37 °C with shaking (#Heatblock). The mixture was centrifuged at 16000 x g for 30 seconds to remove the supernatant. The pellet was resuspended in 50 μl of LB and spread onto a selection plate and incubated overnight at 37°C.

Results:

KLD of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP

Goal:

KLD reaction was performed to assemble constructs … from PCR products … respectively.

Protocol:

KLD reaction was performed using the KLD Enzyme Mix (M0554, New England Biolabs), consisting of a kinase, a ligase, and a DpnI restriction enzyme, according to the manufacturer's instructions. For each reaction, KLD reaction buffer, KLD enzyme mix, nuclease-free water, and the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 μL
KLD Reaction Buffer (2x)	5 μL
KLD Enzyme Mix (10x)	1 μL
Nuclease-free water	3 μL
Total	10 μL

The sample was incubated at room temperature (25 °C) for 5–10 minutes. Optionally, to increase the efficiency of the DpnI digestion, the mixture was additionally incubated at 37 °C for 30–60 minutes. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of KLD reaction was evaluated by downstream experiments.

DNA Ligation for constructs 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES & 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES overnight

Goal:

Ligation of the digested 320_pcDNAT7v2.2 backbone with digested PCRs of @172 & @173 to assemble 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES & 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES.

Protocol:

Ligation was carried out using the T4 DNA Ligase (M0202, New England Biolabs) according to the manufacturers protocol. DNA fragments of interest, including 50 ng of vector with 3-fold molar excess of insert(s), were combined with 2 μL of T4 DNA Ligase Buffer (10x), and 1 μL of nuclease-free water. Molar amount of each DNA fragment was calculated using the NEBioCalculator tool. The sample was mixed by pipetting, and incubated at room temperature for 10 minutes. The reaction was then stopped using heat inactivation at 65°C, 10 minutes.

Component	Volume	600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES	610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES
T4 DNA Ligase Buffer (10x)	2 μL
Vector DNA (320_pcDNAT7v2.2)	50 ng (0.020 pmol)	0.6 µL	0.6 µL
Insert DNA (@172, @173)	42.5 ng (0.060 pmol)	1.5 µL	1.3 µL
Nuclease-free Water	To 20 μL	14.9	15.1
T4 DNA Ligase	1 μL

The reaction was then stored at -20°C or immediately used for transformation (1-5 μL of ligation product).

Results:

The efficiency of restriction digest was evaluated by downstream experiments.

DNA Ligation for constructs 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES & 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES short

Goal:

Ligation of the digested 320_pcDNAT7v2.2 backbone with digested PCRs of @172 & @173 to assemble 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES & 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES.

Protocol:

Ligation was carried out using the T4 DNA Ligase (M0202, New England Biolabs) according to the manufacturers protocol. DNA fragments of interest, including 50 ng of vector with 3-fold molar excess of insert(s), were combined with 2 μL of T4 DNA Ligase Buffer (10x), and 1 μL of nuclease-free water. Molar amount of each DNA fragment was calculated using the NEBioCalculator tool. The sample was mixed by pipetting, and incubated at room temperature for 10 minutes. The reaction was then stopped using heat inactivation at 65°C, 10 minutes.

Component	Volume	600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES	610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES
T4 DNA Ligase Buffer (10x)	2 μL
Vector DNA (320_pcDNAT7v2.2)	50 ng (0.020 pmol)	0.6 µL	0.6 µL
Insert DNA (@172, @173)	42.5 ng (0.060 pmol)	1.5 µL	1.3 µL
Nuclease-free Water	To 20 μL	14.9	15.1
T4 DNA Ligase	1 μL

The reaction was then stored at -20°C or immediately used for transformation (1-5 μL of ligation product).

Results:

The efficiency of restriction digest was evaluated by downstream experiments.

E. coli colony picking of 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal

Colony picking of individual DH10β E. coli (Cat. No. C3019, New England Biolabs) bacterial colonies transformed with … from selection plates containing carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plates. Each colony was transferred to 5 mL of fresh culture medium (LB medium with 100 μg/mL carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm (CERTOMAT BS-1 Incubator Shaker, Sartorius) for further growth and amplification of the selected cells. The agar plates were sealed to prevent contamination and stored at 4 °C.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial cultures after overnight incubation at 37 °C.

Plasmid Miniprep (NEB) of 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4, 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Minipreps of 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4, 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES were performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol:

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight cultures were pelleted by centrifugation for 15 minutes at 4000 ×  g (Eppendorf, Centrifuge 5804R). For each sample, the supernatant was discarded. Pellet was resuspended in 200 μL of plasmid resuspension buffer. Cell lysis was achieved by the addition of 200 μL of plasmid lysis buffer, followed by 5–6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of plasmid neutralization buffer. The tubes were gently inverted and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transferred to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of plasmid wash buffer 1 and centrifuging for 1 minute, followed by 400 μL of plasmid wash buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL Eppendorf tubes. DNA was eluted by the addition of 30 μL of preheated TE buffer (55–65 °C) to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop (#Nano_model), and the DNA samples were stored at −20 °C.

Results:

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
280.1	290,45	1,94	2,49
280.2	227,45	1,94	2,56
280.3	251,40	1,94	2,46
290.1	210,74	1,94	2,55
290.2	244,19	1,94	2,53
290.3	305,93	1,93	2,46
310.1	419,14	1,93	2,41
310.2	346,49	1,93	2,46
310.3	251,77	1,94	2,52
590.1	579,96	1,93	2,34
590.2	383,01	1,94	2,45
590.3	329,21	1,94	2,50
600.1	449,22	1,93	2,37
600.2	363,18	1,94	2,44
600.3	323,21	1,94	2,48
610.1	341,09	1,93	2,43
610.2	362,38	1,94	2,44
610.3	178,32	1,94	2,57

Restriction digest of @171, @172, @173 with HindIII-HF and KpnI-HF

Goal:

Restriction digest of the … with … enzyme(s), for downstream cloning of ….

Protocol:

Restriction digest was carried out using the … enzyme(s) (…,  New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 20 minutes.

Component	Volume
HindIII	0.25 µL
KndI	0.25 µL
rCutSmart buffer	1 µL
DNA	5 µL
Nuclease-free Water	to 10 µL

Optionally, the reaction was stopped using heat inactivation (80 °C, 20 minutes). The products were then stored at –20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

PCR Clean-up (NEB): @172 & @173

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
172	82.9	1.96	5.47
173	95.1	1.97	4.78

Hotstart PCR (Platinum™ SuperFi™ II): 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES & 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from @172 & @173 for constructs 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES with high fidelity and efficiency.

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

label	gBlock	Construct	Primer	length	extension time
2	172	600	146 & 147	1.7 kb	1 min
3	173	610	146 & 147	1.67	1 min

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA	10 ng	1 μL
DMSO (only for 590, 600, 610)	2.5 % (v/v)	0.5 µL
Nuclease-free water	–	6.5 μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	5–10 sec	35
Annealing	60°C	10 sec
Extension	72°C	15–30 seconds/kb
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

Bacterial (Re)Transformation: 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM & 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal(Re-)Transformations are used to insert plasmid 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP,  280_pcDNAT7v1_RS20-cpEFGP-CaM & 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) into bacteria.

Protocol

1. Get a box of ice.
2. Get competent DH5α E. coli cells from -80°C freezer & let it thaw in your hand.
3. Gently mix & pipette 50 µL of cells into a fresh Eppendorf tube which is placed on ice.
4. Add 1-5 µL containing 1 pg-100 ng of plasmid DNA to the cell culture. Carefully flick the tube 4-5 times to mix the cells & DNA. Do not vortex!
5. Place the mixture on ice for 10 min. Do not mix! Warm up heat block in the meantime and get agar plate(s). Plate(s) can be placed in the incubator to dry them for a bit. Plate(s) should be labeled on the bottom (side with the medium) beforehand.
6. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix!
7. Place the tube on ice for 3 minutes. Do not mix!
8. Pipette 200 µL of room temperature SOC into the mixture to recover the cells.
9. Immediately spread 50-100 µL onto a selection plate and spread the liquid evenly.
10. Incubate the plate overnight at 37°C with the lid placed downwards.

ResultThe following day, colonies should have grown and could be picked for further work.

Splitting of HEK293T cell line into T25 flask - p21

Goal:

Splitting HEK293t cell line from "HEK" flask  to the next passage number of p21.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Culturing media was decanted from the 25 cm 2 culturing flask. Cells were washed using 2.5 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 2 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos's modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 25 cm 2 flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO 2 and 90% relative humidity.

Results:

HEK293t cell line from "HEK" flask  to the next passage number of p21.

Splitting of HEK293T cell line into T25 flask - p22

Goal:

Splitting HEK293t cell line from "HEK" flask  to the next passage number of p22.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Culturing media was decanted from the 25 cm 2 culturing flask. Cells were washed using 2.5 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 2 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos's modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 25 cm 2 flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO 2 and 90% relative humidity.

Results:

HEK293t cell line from "HEK" flask  to the next passage number of p22.

Touchdown PCR (Platinum™ SuperFi™ II) of @153, @154, 200_pcDNAT7v1

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from @153, @154, and 200_pcDNAT7v1 for constructs 280_pcDNAT7v1_RS20-cpEFGP-CaM and 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) with high fidelity and efficiency.

	gBlock	Primer	length	extension time
280	153	282 & 281	1.3 kb	50 sec
	200	254 & 283	6 kb	3 min 15 sec
290	154	281 & 292	1.5 kb	50 sec
	200	293 & 254	6 kb	3 min 15 sec

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA	10 ng	1 μL
DMSO	2.5 %	0.5 µL
Nuclease-free water	–	6.5 μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

	Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Touchdown phase	Denaturation	98°C	10 sec	10
Annealing	start: 67°C (every cycle -1 °C) end: 57 °C	20 sec
Extension	72°C	15–30 seconds/kb
plateau phase	Denaturation	98 °C	10 sec	25
Annealing	57 °C	20 sec
Extension	72 °C	15 - 30 sec/kb
	Final extension	72°C	5 min	1
	Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

PCR (Platinum™ SuperFi™ II) of 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP for constructs 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP with high fidelity and efficiency.

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each) 271 & 272	0.5 μM	2 μL
Template DNA (250.4)	10 ng	1 μL
DMSO	2.5 %	0.5 µL
Nuclease-free water	–	6.5 μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	10 sec	35
Annealing	58°C	20 sec
Extension	72°C	4 min  30 sec
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

E. coli colony picking for 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal

Colony picking of individual DH10β E. coli (Cat. No. C3019, New England Biolabs) bacterial colonies transformed with 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES from selection plates containing carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol

Using a sterile inoculation loop or pipette tip, 3 individual colonies were carefully picked from the surface of each selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with 100 μg/mL carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm (CERTOMAT BS-1 Incubator Shaker, Sartorius) for further growth and amplification of the selected cells. The agar plates were sealed to prevent contamination and stored at 4 °C.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial cultures after overnight incubation at 37 °C.

Agarose Gel Electrophoresis of analytical digestion of 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Protocol:

Agarose gel electrophoresis was performed using a 1% w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5–10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right: ladder, 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES.1, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES.1, 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES.2 Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

Splitting of HEK293T cell line into T25 flask - p20

Goal:

Splitting HEK293t cell line from "HEK" flask  to the next passage number of p20.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Culturing media was decanted from the 25 cm 2 culturing flask. Cells were washed using 2.5 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 2 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 4 ml pre-warmed complete Dulbeccos's modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 25 cm 2 flask was inoculated with the 1ml cell suspension to 5 ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO 2 and 90% relative humidity.

Results:

Splitting HEK293t cell line from "HEK" flask  to the next passage number of p20.

Purification of 330_LET_CLS-XaCS-BmTyr using Strep-Tactin(R)XT 4 Flow(R) high capacity Spin Column Kit

Purification using Strep-Tactin(R)XT 4 Flow(R) high capacity Spin Column Kit

performed to purify small amount of Strep-tag®II or Twin-Strep-tag® protein ....

• Resuspend Strep-Tactin®XT 4Flow® high capacity resin and pipet 100 μl of the 50% suspension into a spin column leading to a column bed volume of 50 μl
• Centrifuge the sample (maximum speed, 5 min, 4 °C) to remove aggregates
• Apply up to 500 μl sample to the spin column, close the column lid and incubate at room temperature with constant movement (rolling or shaking) for 5-30 min. For most purposes, 5 min are sufficient, but a longer incubation can increase the amount of captured protein, especially for large proteins (> 90 kDa)
• After incubation, open the lid and break of the lower column seal. Place the spin column into a reaction tube and centrifuge for 30 sec at 500 x g
• Collect the flow-through for SDS-PAGE analysis and place the spin column into a new reaction tube. Apply 500 μl 1x Buffer W and centrifuge for 30 seconds at 700 x g. (Optional: Usually, one washing step is sufficient to obtain a highly pure protein, but this step can be repeated if higher purity is required.)
• Collect the washing fraction for SDS-PAGE analysis and place the spin column into a new reaction tube. Proceed with elution step a or b:

a. For fast processing, apply 150-200 μl 1x Buffer BXT, close the spin column lid and vortex briefly. After 5 minutes of incubation without movement, vortex again briefly, open the spin column lid and centrifuge for 30 seconds at 700 x g.b. For maximum target protein concentration, apply 50-100 μl 1x Buffer BXT, close the spin column lid and vortex briefly. After 5 minutes of incubation without movement, vortex again briefly, open the spin column lid and centrifuge for 30 seconds at 700 x g. Repeat this step one to two times. At least 80% of the target protein will be in the first elution fraction.

Analytical digest of 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 repeated

Goal:

Restriction digest of the 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 minipreps with restriction enzymes. 280.1, 280.2, 290.1, 290.2, 310.2, 310.3 minipreps with Mlul HF enzyme for checking purposes.

Construct	280	290	310	590	600	610
Enzyme 1	KpnI	ApaI	BsmBI	KpnI	KpnI	KpnI
Enzyme 2	MIuI	MIuI	MIuI	HindIII	HindIII	HindIII
Fragments	6 kb & 1.3 kb		6 kb & 0.7 kb	6 kb & 1.7 kb	6 kb & 1.7 kb	6 kb & 1.7 kb

Due to different enzyme stock in the lab ApaI was not used, instead a corresponding amount of MIuI was added and later at the gel the overall length was checked.

Protocol:

Restriction digest was carried out using the KpnI enzyme (R3142,  New England Biolabs) and MluI enzyme (R3198, New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 1 μL of rCutSmart buffer (B7204, New England Biolabs) and 1 U of the respective enzymes in a PCR tube with a total reaction volume of 10 μL.

Master mix for the digestion of 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES was prepared as follows:

Component	Volume
Restriction enzyme HindIII	2.5 μL
Restriction enzyme KpnI	2.5 µL
rCutSmart buffer	10 μL
Nuclease-free Water	43 μL

Master mix for the digestion of 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4:

Component	Volume
Restriction enzyme MIuI	1 μL
Restriction enzyme 2 (KpnI, ApaI, or BsmBI)	1 µL
rCutSmart buffer	10 μL
Nuclease-free Water	18 μL

Due to different enzyme stock in the lab ApaI was not used, instead a corresponding amount of MIuI was added and later at the gel the overall length was checked.

5 μl of the master mix was supplemented with 5 μl of template DNA for the digest. The samples were mixed by pipetting and incubated at 37 °C for 20 minutes.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

PCR (Platinum™ SuperFi™ II): 330_LET_CLS-XaCS-BmTyr, 340_LET_CLS-XaCS-BmTyr(E195S+A221V), 350_LET_CLS-XaCS-BmTyr(R209S+M215E+V218M), 360_LET_CLS-XaCS-BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C), 370_LET_CLS-XaCS-cpBmTyr(201), 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201), 440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466), 460_LET_CLS-XaCS-LsTyr, 470_LET_CLS-XaCS-SkMelC1(33-124), 480_LET_CLS-XaCS-SkMelC2, 490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518), 510_LET_CLS-XaCS-SavMelC1(27-118), 520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F), 530_LET_CLS-XaCS-SavMelC2 and 540_LET_CLS-XaCS-SavMelC2(I42Y)

Goal:

Polymerase chain reaction (PCR) was performed to linearizise the specific DNA fragments 330_LET_CLS-XaCS-BmTyr, 340_LET_CLS-XaCS-BmTyr(E195S+A221V), 350_LET_CLS-XaCS-BmTyr(R209S+M215E+V218M), 360_LET_CLS-XaCS-BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C), 370_LET_CLS-XaCS-cpBmTyr(201), 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201), 440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466), 460_LET_CLS-XaCS-LsTyr, 470_LET_CLS-XaCS-SkMelC1(33-124), 480_LET_CLS-XaCS-SkMelC2, 490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518), 510_LET_CLS-XaCS-SavMelC1(27-118), 520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F), 530_LET_CLS-XaCS-SavMelC2 and 540_LET_CLS-XaCS-SavMelC2(I42Y) with high fidelity and efficiency.

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each) - 146/147	0.5 μM	2 μL
Template DNA	variable	2 μL
Nuclease-free water	–	6 μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	10 sec	32
Annealing	60°C	10 sec
Extension	72°C	30 seconds
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

Analytical digest of 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4, 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal:

Restriction digest of the 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4, 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES minipreps with and enzymes, as well as 290.3, 290.4, 290.5, and 290.6 minipreps with Mlul HF enzyme for checking purposes.

Construct	280	290	310	590	600	610
Enzyme 1	KpnI	ApaI	BsmBI	KpnI	KpnI	KpnI
Enzyme 2	MIuI	MIuI	MIuI	HindIII	HindIII	HindIII
Fragments	6 kb & 1.3 kb		6 kb & 0.7 kb	6 kb & 1.7 kb	6 kb & 1.7 kb	6 kb & 1.7 kb

Protocol:

Restriction digest was carried out using the KpnI enzyme (R3142,  New England Biolabs) and MluI enzyme (R3198, New England Biolabs) according to the manufacturer's instructions. Template DNA was supplied in an amount of 1 μg and combined with 1 μL of rCutSmart buffer (B7204, New England Biolabs) and 1 U of the respective enzymes in a PCR tube with a total reaction volume of 10 μL.

Master mix for the digestion of 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES was prepared as follows:

Component	Volume
Restriction enzyme HindIII	2.5 μL
Restriction enzyme KpnI	2.5 µL
rCutSmart buffer	10 μL
Nuclease-free Water	43 μL

Master mix for the digestion of 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4:

Component	Volume
Restriction enzyme MIuI	1 μL
Restriction enzyme 2 (KpnI, ApaI, or BsmBI)	1 µL
rCutSmart buffer	10 μL
Nuclease-free Water	18 μL

5 μl of the master mix was supplemented with 5 μl of template DNA for the digest. The samples were mixed by pipetting and incubated at 37 °C for 20 minutes.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

E. coli colony picking of 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4, 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES

Goal

Colony picking of individual DH10β E. coli (Cat. No. C3019, New England Biolabs) bacterial colonies transformed with 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4, 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES from selection plates containing carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plates. Each colony was transferred to 5 mL of fresh culture medium (LB medium with 100 μg/mL carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm (CERTOMAT BS-1 Incubator Shaker, Sartorius) for further growth and amplification of the selected cells. The agar plates were sealed to prevent contamination and stored at 4 °C.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial cultures after overnight incubation at 37 °C.

E. coli colony picking of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal

Colony picking of individual DH10β E. coli (Cat. No. C3019, New England Biolabs) bacterial colonies transformed with 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 from selection plates containing carbenicillin was performed for the replication and expression of the desired genetic material. 6 colonies were selected per construct.

Protocol

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plates. Each colony was transferred to 5 mL of fresh culture medium (LB medium with 100 μg/mL carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm (CERTOMAT BS-1 Incubator Shaker, Sartorius) for further growth and amplification of the selected cells. The agar plates were sealed to prevent contamination and stored at 4 °C.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial cultures after overnight incubation at 37 °C.

Plasmid Maxiprep (QIAGEN) of 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP and 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP

Goal:

A maxiprep of 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP and 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Protocol:

DNA plasmids were prepared using the QIAGEN ® Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). ... mL of overnight culture of E. coli was centrifuged at 4500 × g for 20 min at 4°C. The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer with thorough mixing. The mixture was centrifuged at 7000 × g (eppendorf 5430 R) for 135 min (if supernatant not clear, optional +30) min at 4 °C. The supernatant was applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuging at 7000 × g for 75 min (if pellet not strong enough, additional 30 min) at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol. Finally, the supernatant was aspirated using a vacuum pump without disturbing the pellet, and the pellet was air-dried for 5 - 10 min. The DNA was then redissolved in 200 μL of TE buffer, the concentration was determined using a NanoDrop, and the DNA was stored at −20 °C.

Results:

The maxiprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
250.4	857.68	1.95	2.24
250.5	2065.17	1.96	2.24
260.3	2692.27	1.94	2.23

Sanger sequencing of 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP and 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP

Goal:

To confirm the nucleotide sequences of …, Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a (partial) match between the experimental and expected sequences. In addition, the sequencing data showed (predominantly) a high quality score and a continuous read length of over 1 kb.

Sample	Barcode	Primer	Validation
250.4		CMV-fw
250.4		WPRE-rev
250.5		CMV-fw
250.5		WPRE-rev
260.3		CMV-fw
260.3		WRPE-rev

PCR Clean-up (NEB) of digested @171, @172, @173

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
171	37.91	1.81	10.37
172	39.20	1.86	6.09
173	44.79	1.90	9.83

Gibson Assembly of 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

Gibson assembly was performed to assemble the 200_pcDNAT7v1 vector and @153, @154, and @155 inserts to produce 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4.

Protocol:

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Components	Volume	280	290	310
NEBuilder HiFi DNA Assembly Master Mix	10 µL	10 µL	10 µL	10 µL
Vector	… ng	3.8 µL	3.2 µL	3.4 µL
Insert	… ng	0.6 µL	0.8 µL	0.6 µL
Nuclease-free Water	... μL	5.6 µL	5.8 µL	6.1 µL
Total	20 µL	20 µL	20 µL	20 µL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of Gibson assembly was evaluated by downstream experiments.

Amplification of DNA using the phi29-XT RCA Kit for purified circular DNA 200_pcDNAT7v1 and 320_pcDNAT7v2

Goal:

DNA of 200_pcDNAT7v1 was amplified using the phii29-XT RCA kit (NEB #E1603) for downstream applications.

Protocol:

Reactions with an end volume of 20 µL were prepared without enzyme as described in the table below. For each reaction, 4 µL of phi29-XT Reaction Buffer (5X) was added to achieve a final concentration of 1X, as well as 2 µL of Deoxynucleotide (dNTP) Solution Mix (10 mM) for a final concentration of 1 mM.  Furthermore, 2 µL of Exonuclease-Resistant Random Primers (500 µM) were added to achieve a final concentration of 50 µM, as well as X µL of the  DNA template for a final concentration of X. Nuclease-free water was used to bring the reaction to 18 µL.

Components	20 µL reaction	Final concentration	pA	pB	pC
phi29-XT Reaction Buffer, 5X	4 µL	1X	4 µL	4 µL	4 µL
Deoxynucleotide (dNTP) Solution Mix, 10 mM	2 µL	1 mM	2 µL	2 µL	2 µL
Exonuclease-Resistant Random Primers, 500 µM	2 µL	50 µM	2 µL	2 µL	2 µL
Circular DNA Template	1 µL	100 pg	1 µL (320_pcDNAT7v2)	1 µL (320_pcDNAT7v2)	1 µL (200_pcDNAT7v1)
Nuclease-free Water	9 µL	N/A	9 µL	9 µL	9 µL
42 °C Incubation time			2 h	4 h	4 h

Each reaction was mixed thoroughly by gentle pipetting or vortexing, followed by brief centrifuging.

After that, the reactions were incubated in a thermocycler (model) at 95 °C for 3 minutes, with a lid temperature of 100 °C, followed by cooling the reactions at room temperature.

The reactions were put on ice, and 2 µL of phi29-XT DNA Polymerase was added to each reaction, followed by mixing by gentle pipetting or vortexing, and brief centrifuging.

Lastly, the reactions were incubated in a thermocycler (model) at 42 °C for pA) 320_pcDNAT7v2, 2 hours; for pB) 320_pcDNAT7v2, 4 hours; pC) 200_pcDNAT7v1, 4 hours, with a lid temperature of ≥ 75°C, followed by 10 minutes at 65 °C to inactivate the DNA polymerase.

The RCA products can be kept at 4 °C overnight or at  -20 °C for long-term storage.

Results:

DNA Ligation: 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M), 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)

Goal:

Ligation of the 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M) and 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M).

Protocol:

Ligation was carried out using the T4 DNA Ligase (M0202, New England Biolabs) according to the manufacturers protocol. DNA fragments of interest, including 50 ng of vector with 2 μL of T4 DNA Ligase Buffer (10x), and 1 μL of nuclease-free water. Molar amount of each DNA fragment was calculated using the NEBioCalculator tool. The sample was mixed by pipetting, and incubated at 16°C for 12 hours, followed by heat inactivation at 65°C for 10 minutes.

Component	Volume 20 µL
T4 DNA Ligase Buffer (10x)	2 μL
Vector DNA	50 ng (0.020 pmol)
Nuclease-free Water	To 20 μL
T4 DNA Ligase	1 μL

The reaction was then stored at -20°C or immediately used for transformation (1-5 μL of ligation product).

Results:

The efficiency of restriction digest was evaluated by downstream experiments.

E. coli transformation of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 v2 & v3

Goal:

Transformation was performed to introduce plasmid DNA constructs 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 into DH10β E. coli cells, allowing for the replication and expression of the desired genetic material. Two versions of each construct were transformed.

Protocol:

For each construct, an aliquot of competent DH10β E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells (1–5 μL) and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 μL of room temperature SOC, the mixture was then recovered for 30–60 min at 37 °C with shaking (#Heatblock). The mixture was centrifuged at 16000 x g for 30 seconds to remove the supernatant. The pellet was resuspended in 50 μl of LB and spread onto a selection plate and incubated overnight at 37°C.

Results:

Splitting of HEK293T cell line into T25 flask - p18

Goal:

Splitting HEK293t cell line from "I", "II", "III", "IV-PS" and "V-PS" flasks to the next passage number of p18.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Culturing media was decanted from the 25 cm^2 culturing flask. Cells were washed using 2.5 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 2 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 4 ml pre-warmed complete Dulbeccos's modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. The same 25 cm^2 flask was inoculated with the 1ml cell suspension to 5 ml pre-warmed DMEM. 1% PenStrep was additionally added to "IV" and "V" flasks. Cells were grown at 37°C with 5% CO 2 and 90% relative humidity.

Results:

HEK293t cell line from "I", "II", "III", "IV-PS" and "V-PS" flask was split to passage number p18.

Preparation of LB agar plates

Goal

Protocol

A bottle containing sterile LB agar was heated in the microwave until fully liquefied. After cooling to hand-warm temperature, the appropriate antibiotic (carbenicillin to the final concentration of 100 μg/mL) was added. Plates were poured under the laminar flow hood and allowed to dry (~15 mins).

Results

Plates were successfully prepared and stored in the cold room for downstream applications.

Splitting of HEK293T cell line into T25 flask - p17

Goal:

Splitting HEK293t cell line from "I", "II", "III", "IV-PS" and "V-PS" flasks to the next passage number of p17.

Protocol:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work. Culturing media was decanted from the 25 cm^2 culturing flask. Cells were washed using 2.5 ml of pre-warmed Dulbecco's phosphate-buffered saline (DPBS) and detached using 2 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 4 ml pre-warmed complete Dulbeccos's modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. The same 25 cm^2 flask was inoculated with the 1ml cell suspension to 5 ml pre-warmed DMEM. 1% PenStrep was additionally added to "IV" and "V" flasks. Cells were grown at 37°C with 5% CO 2 and 90% relative humidity.

Results:

HEK293t cell line from "I", "II", "III", "IV-PS" and "V-PS" flask was split to passage number p17.

PCR (Platinum™ SuperFi™ II) of 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, and 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from @171, @172, and @173 for constructs 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES with high fidelity and efficiency.

gBlock	Construct	Primer	length	extension time
171	590	146 & 147	1.7 kb	1 min
172	600	146 & 147	1.7 kb	1 min
173	610	146 & 147	1.67	1 min

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA	10 ng	1 μL
DMSO	2.5 % (v/v)	0.5 µL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	5–10 sec	35
Annealing	58°C	20 sec
Extension	72°C	30 seconds/kb
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

PCR Clean-up (NEB) of digested 320_pcDNAT7v2

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of 2 × 10 µl of 60°C TE buffer to the column, with 5 mins of incubation time. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
320.1	79.5	1.90	3.57
320.2	62.7	1.93	3.74

Restriction digest of 320_pcDNAT7v2, @171, @172, and @173

Goal:

Restriction digest of 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4, @171, @172, and @173 with KpnI and HindIII enzyme(s), for downstream cloning of 590_pcDNAT7v2_3xLBT15-TEVCS(M)-MxEnc-eUnaG-STII-NES, 600_pcDNAT7v2_3xLBT15-TEVCS(M)-QtEnc-eUnaG-STII-NES, and 610_pcDNAT7v2_3xLBT15-TEVCS(M)-TmEnc-eUnaG-STII-NES.

Protocol:

Restriction digest was carried out using the KpnI and HindIII according to the manufacturer's instructions. For the inserts, template DNA was supplied in an amount of 5 μg and combined with 10 μL of rCutSmart (B7204, New England Biolabs) and 5 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 1 hour.For the backbone, template DNA was supplied in an amount of 5 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 5 μL of the respective enzymes in a PCR tube. The samples were mixed by pipetting and incubated at 37 °C for 2 hours.

Component	Volume	320_pcDNAT7v2
Restriction enzyme KpnI	10 U (1 μL)	50 U (5 µL)
Restriction enzyme HindIII	10 U (1 µL)	50 U (5 µL)
rCutSmart buffer	5 μL	10 µL
DNA of @171, @172, and @173	1 μg	5 µg
Nuclease-free Water	to 50 μL	to 100 µL

Sample 320_pcDNAT7v2 was dephosphorylated using CIP (number, NEB) by adding 7.5µl of CIP to the digest. After 1 hour of CIP, the digest was stopped.

The digest products were cleaned with the NEB PCR Clean-up kit.

The products were then stored at –20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

PCR (Platinum™ SuperFi™ II): linearisation 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M) and 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M) and 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M) with high fidelity and efficiency.

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA	variable	2 μL
Nuclease-free water	–	6 μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	10 sec	32
Annealing	60°C	10 sec
Extension	72°C	1min 20 sec
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

Gibson Assembly of @156 for 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M) and 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)

Goal:

Gibson assembly was performed to assemble the @156 vector and PCR1 insert to produce 440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466) and @156 vector and PCR2 insert to produce 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M).

Protocol:

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Components	Volume
NEBuilder HiFi DNA Assembly Master Mix	10 µL
Vector	20 ng
Insert	20 ng
Nuclease-free Water	x μL
Total	20 µL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of Gibson assembly was evaluated by downstream experiments.

DNA Ligation: 330_LET_CLS-XaCS-BmTyr, 340_LET_CLS-XaCS-BmTyr(E195S+A221V), 350_LET_CLS-XaCS-BmTyr(R209S+M215E+V218M), 360_LET_CLS-XaCS-BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C), 370_LET_CLS-XaCS-cpBmTyr(201), 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201), 440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466), 460_LET_CLS-XaCS-LsTyr, 470_LET_CLS-XaCS-SkMelC1(33-124), 480_LET_CLS-XaCS-SkMelC2, 490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518), 510_LET_CLS-XaCS-SavMelC1(27-118), 520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F), 530_LET_CLS-XaCS-SavMelC2 and 540_LET_CLS-XaCS-SavMelC2(I42Y)

Goal:

Ligation of the 330_LET_CLS-XaCS-BmTyr, 340_LET_CLS-XaCS-BmTyr(E195S+A221V), 350_LET_CLS-XaCS-BmTyr(R209S+M215E+V218M), 360_LET_CLS-XaCS-BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C), 370_LET_CLS-XaCS-cpBmTyr(201), 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201), 440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466), 460_LET_CLS-XaCS-LsTyr, 470_LET_CLS-XaCS-SkMelC1(33-124), 480_LET_CLS-XaCS-SkMelC2, 490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518), 510_LET_CLS-XaCS-SavMelC1(27-118), 520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F), 530_LET_CLS-XaCS-SavMelC2 and 540_LET_CLS-XaCS-SavMelC2(I42Y).

Protocol:

Ligation was carried out using the T4 DNA Ligase (M0202, New England Biolabs) according to the manufacturers protocol. DNA fragments of interest, including 50 ng of vector with 2 μL of T4 DNA Ligase Buffer (10x), and 1 μL of nuclease-free water. Molar amount of each DNA fragment was calculated using the NEBioCalculator tool. The sample was mixed by pipetting, and incubated at 16°C for 12 hours, followed by heat inactivation at 65°C for 10 minutes.

Component	Volume 20 µL
T4 DNA Ligase Buffer (10x)	2 μL
Vector DNA	50 ng (0.020 pmol)
Nuclease-free Water	To 20 μL
T4 DNA Ligase	1 μL

The reaction was then stored at -20°C or immediately used for transformation (1-5 μL of ligation product).

Results:

The efficiency of restriction digest was evaluated by downstream experiments.

PCR Clean-up (NEB): 330_LET_CLS-XaCS-BmTyr, 340_LET_CLS-XaCS-BmTyr(E195S+A221V), 350_LET_CLS-XaCS-BmTyr(R209S+M215E+V218M), 360_LET_CLS-XaCS-BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C), 370_LET_CLS-XaCS-cpBmTyr(201), 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201), 440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466), 460_LET_CLS-XaCS-LsTyr, 470_LET_CLS-XaCS-SkMelC1(33-124), 480_LET_CLS-XaCS-SkMelC2, 490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518), 510_LET_CLS-XaCS-SavMelC1(27-118), 520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F), 530_LET_CLS-XaCS-SavMelC2 and 540_LET_CLS-XaCS-SavMelC2(I42Y)

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g . The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 ×  g . DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
330	154.04	1.91	2.13
340	133.80	1.89	2.08
350	145.18	1.92	2.48
360	121.86	1.91	3.31
370	141.83	1.89	3.09
380	83.24	1.88	2.18
390	199.23	1.92	2.82
400	206.11	1.92	2.74
410	135.40	1.91	3.37
420	156.01	1.90	2.03
430	193.44	1.90	2.13
440	153.21	1.90	2.66
460	225.56	1.91	2.09
470	149.64	1.89	3.21
480	104.67	1.90	4.09
490	93.09	1.86	4.71
510	181.36	1.90	2.15
520	167.25	1.92	2.11
530	13.48	1.62	1.08
540	138.09	1.89	1.99

Sanger sequencing of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

To confirm the nucleotide sequences of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4, Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a (partial) match between the experimental and expected sequences. In addition, the sequencing data showed (predominantly) a high quality score and a continuous read length of over 1 kb.

Sample	Barcode	Primer	Validation
270.a3	06817507	T7	No insert
270.a3	06817508	T7-Term	No insert
270.a4	06817509	T7	No insert
270.a4	06817510	T7-Term	No insert
270.a5	06817511	T7	No insert
270.a5	06817512	T7-Term	No insert
270.a6	06817513	T7	No insert
270.a6	06817514	T7-Term	No insert
310.6	06817515	T7	No insert
310.6	06817515	T7-Term	No insert
310.7	06817517	T7	No insert
310.7	06817518	T-Term	No insert

Plasmid Miniprep (NEB) 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP 280_pcDNAT7v1_RS20-cpEFGP-CaM 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

Minipreps of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 were performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol:

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight cultures were pelleted by centrifugation for 15 minutes at 4000 ×  g (Eppendorf, Centrifuge 5804R). For each sample, the supernatant was discarded. Pellet was resuspended in 200 μL of plasmid resuspension buffer. Cell lysis was achieved by the addition of 200 μL of plasmid lysis buffer, followed by 5–6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of plasmid neutralization buffer. The tubes were gently inverted and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transferred to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of plasmid wash buffer 1 and centrifuging for 1 minute, followed by 400 μL of plasmid wash buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL Eppendorf tubes. DNA was eluted by the addition of 30/100 μL of preheated TE buffer (55–65 °C) to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop (#Nano_model), and the DNA samples were stored at −20 °C.

Results:

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range. Note: some are eluted in 100 uL some are eluted in 30

Sample	Concentration [ng/μL]	A260/280	A260/230	Elution Buffer Volume
270c.1	601.49	1.92	2.24	30 uL
270c.2	573.23	1.93	2.30	30 uL
270c.3	525.39	1.93	2.30	30 uL
270c.4	554.92	1.93	2.29	30 uL
270c.5	206.11	1.94	2.29	100 uL
270c.6	175.23	1.94	2.28	100 uL
280c.1	634.13	1.95	2.29	30 uL
280c.2	548.31	1.92	2.30	30 uL
280c.3	191.20	1.94	2.28	100 uL
280c.4	183.29	1.95	2.28	100 uL
280c.5	177.84	1.93	2.24	100 uL
280c.6	222.49	1.94	2.29	100 uL
290c.1	222.26	1.95	2.28	100 uL
290c.2	510.18	1.92	2.32	30 uL
290c.3	510.44	1.93	2.30	30 uL
290c.4	459.46	1.91	2.46	30 uL
290c.5	553.67	1.92	2.27	30 uL
290c.6	493.07	1.94	2.32	30 uL
310c.1	518.22	1.93	2.30	30 uL
310c.2	453.32	1.92	2.36	30 uL
310c.3	546.42	1.94	2.33	30 uL
310c.4	179.68	1.96	2.27	100 uL
310c.5	482.58	1.93	2.29	30 uL
310c.6	158.58	1.93	2.37	100 uL

Plasmid Miniprep (NEB) of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP

Goal:

Minipreps of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP were performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol:

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight cultures were pelleted by centrifugation for 15 minutes at 4000 ×  g (Eppendorf, Centrifuge 5804R). For each sample, the supernatant was discarded. Pellet was resuspended in 200 μL of plasmid resuspension buffer. Cell lysis was achieved by the addition of 200 μL of plasmid lysis buffer, followed by 5–6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of plasmid neutralization buffer. The tubes were gently inverted and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transferred to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of plasmid wash buffer 1 and centrifuging for 1 minute, followed by 400 μL of plasmid wash buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL Eppendorf tubes. DNA was eluted by the addition of 30 μL of preheated TE buffer (55–65 °C) to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop (#Nano_model), and the DNA samples were stored at −20 °C.

Results:

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
270a.3	711.7	1.91	2.37
270a.4	685.5	1.91	2.37
270a.5	709.8	1.90	2.34
270a.6	718.8	1.90	2.34

Sanger sequencing of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP RCA samples

Goal:

To confirm the nucleotide sequences of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a (partial) match between the experimental and expected sequences. In addition, the sequencing data showed (predominantly) a high quality score and a continuous read length of over 1 kb.

Sample	Barcode	Primer	Validation
270a.3	06817499	T7	No priming
270a.3	06817500	T7-Term	No priming
270a.4	06817501	T7	No priming
270a.4	06817502	T7-Term	No priming
270a.5	06817503	T7	No priming
270a.5	06817504	T7-Term	No priming
270a.6	06817505	T7	No priming
270a.6	06817506	T7-Term	No priming

Plasmid Miniprep (NEB) 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

A miniprep of 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol:

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 1 minutes at 16000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5–6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. Final dry centrifugation at 16000 × g for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of prewarmed TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop (#Nano_model), and the DNA samples were stored at 4 °C.

Results:

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
210.2	180.7	1.95	2.75
210.3	156.78	1.97	2.65
250.6	148.21	1.93	2.6
250.7	508.97	1.94	2.35
260.5	112.47	1.97	2.76
260.6	203.92	1.94	2.51
290.3	76.87	1.92	2.49
290.4	51.01	1.91	3.83
310.4	86.56	1.94	3.07
310.5	9.32	1.82	-1.51

Sanger sequencing of 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

To confirm the nucleotide sequences of 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4, Sanger sequencing was performed by using Genewiz, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 50 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis ... a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Sample	Barcode	Primer	Validation
210.2	06817440	T7	Success
210.2	06817441	T7-Term	Success
210.3	06817442	T7	Success
210.3	06817443	T7-Term	Success
250.6	06817444	T7	Missing insert
250.6	06817445	T7-Term	Missing insert
250.7	06817446	T7	Missing insert
250.7	06817447	T7-Term	Missing insert
260.5	06817448	T7	Misssense point mutation in the CDS
260.5	06817449	T7-Term	Misssense point mutation in the CDS
260.6	06817450	T7	Success
260.6	06817451	T7-Term	Success
290.3	06817452	T7	Missing insert
290.3	06817453	T7-Term	Missing insert
290.4	06817454	T7	Missing insert
290.4	06817455	T7-Term	Missing insert
310.4	06817456	T7	Missing insert
310.4	06817457	T7-Term	Missing insert

Sanger sequencing 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

To confirm the nucleotide sequences of 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 Sanger sequencing was performed by using Genewiz, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30–100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis ... a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Sample	Barcode	Primer	Validation
250.4	06817424	T7	Success
250.4	06817425	T7 Term	Success
250.5	06817426	T7	Success
250.5	06817427	T7 Term	Success
260.4	06817428	T7	No insert
260.4	06817429	T7 Term	No insert
280.2	06817430	T7	Deletion inside the integrated region
280.2	06817431	T7 Term	Deletion inside the integrated region
280.3	06817432	T7	Deletion inside the integrated region
280.3	06817433	T7 Term	Deletion inside the integrated region
290.2	06817434	T7	No insert
290.2	06817435	T7 Term	No insert
310.2	06817436	T7	Deletion inside the integrated region
310.2	06817437	T7 Term	Deletion inside the integrated region
310.3	06817438	T7	Deletion inside the integrated region
310.3	06817439	T7 Term	Deletion inside the integrated region

Plasmid Miniprep (NEB) 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

Minipreps of 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 were performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol:

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight cultures were pelleted by centrifugation for 15 minutes at 4000 ×  g (Eppendorf, Centrifuge 5804R). For each sample, the supernatant was discarded. Pellet was resuspended in 200 μL of plasmid resuspension buffer. Cell lysis was achieved by the addition of 200 μL of plasmid lysis buffer, followed by 5–6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of plasmid neutralization buffer. The tubes were gently inverted and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transferred to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of plasmid wash buffer 1 and centrifuging for 1 minute, followed by 400 μL of plasmid wash buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL Eppendorf tubes. DNA was eluted by the addition of 30 μL of preheated TE buffer (55–65 °C) to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop (#Nano_model), and the DNA samples were stored at −20 °C.

Results:

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
250.4	301.69	1.91	2.18
250.5	66.28	1.79	1.70
260.4	450.70	1.93	2.19
280.2	455.33	1.92	2.17
280.3	457.39	1.91	2.17
290.2	459.98	1.90	2.16
310.2	566.67	1.90	2.16
310.3	360.73	1.91	2.17

Gibson Assembly of 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201)

Goal:

Gibson assembly was performed to produce 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, and 430_LET_CLS-XaCS-P3-CBmTyr(201).

Protocol:

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - fold molar excess of insert(s), were combined with 10 μL of master mix.

Components	380_LET_CLS-XaCS-NBmTyr(85)-P4	390_LET_CLS-XaCS-P3-CBmTyr(85)	400_LET_CLS-XaCS-NBmTyr(157)-P4	410_LET_CLS-XaCS-P3-CBmTyr(157)	420_LET_CLS-XaCS-NBmTyr(201)-P4	430_LET_CLS-XaCS-P3-CBmTyr(201)
NEBuilder HiFi DNA Assembly Master Mix	10 µL	10 µL	10 µL	10 µL	10 µL	10 µL
Vector	0.2 µL	0.6 µL	0.2 µL	0.6 µL	0.2 µL	0.6 µL
Insert	0.15 µL	0.2 µL	0.3 µL	0.2 µL	0.3 µL	0.15 µL
Nuclease-free Water	9.65 μL	9.2 µL	9.5 µl	9.2 µL	9.5 µL	9.25 µL
Total	20 µL	20 µL	20 µL	20 µL	20 µL	20 µL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of Gibson assembly was evaluated by downstream experiments.

Plasmid Maxiprep (QIAGEN) of 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)

Goal:

A maxiprep of 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K) was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Protocol:

DNA plasmids were prepared using the QIAGEN ® Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). ... mL of overnight culture of E. coli was centrifuged at 4500 × g for 20 min at 4°C. The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer with thorough mixing. The mixture was centrifuged at 7000 × g (eppendorf 5430 R) for 135 min (if supernatant not clear, optional +30) min at 4 °C. The supernatant was applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuging at 7000 × g for 75 min (if pellet not strong enough, additional 30 min) at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol. Finally, the supernatant was aspirated using a vacuum pump without disturbing the pellet, and the pellet was air-dried for 5 - 10 min. The DNA was then redissolved in 200 μL of TE buffer, the concentration was determined using a NanoDrop, and the DNA was stored at −20 °C.

Results:

The maxiprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
250.2	4774	1.83	2.06
230.a1	2193	1.93	2.26

PCR (Platinum™ SuperFi™ II) for @156, @157, @162 and @166 for constructs 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201), 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M), 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M), LET-P3, and P4-LET

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from @156, @157, @162 and @166 for constructs 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201), 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M), 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M), LET-P3, and P4-LET with high fidelity and efficiency.

Protocol:

Nr. during cloning process	Template	Forward primer	Reverse primer	Cloning intermediate	Expected length
1	@162	142_F_5'LET	452_R_HcTyr1(1-296)-TEVCS(M)_3'LET	450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)	1,353
2	@166	142_F_5'LET	502_R_VsTyr(37-357)-TEVCS(M)_3'LET	500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)	1,500
3	@156	144_F_3'LET	392_R_5'LET_P3	390_LET_CLS-XaCS-P3-CBmTyr(85), 410_LET_CLS-XaCS-P3-CBmTyr(157), 430_LET_CLS-XaCS-P3-CBmTyr(201)	940
4	@156	381_F_3'LET_P4	145_R_5'LET	380_LET_CLS-XaCS-NBmTyr(85)-P4, 400_LET_CLS-XaCS-NBmTyr(157)-P4, 420_LET_CLS-XaCS-NBmTyr(201)-P4	940
5	@157	142_F_5'LET	382_R_NBmTyr(85)_P4	380_LET_CLS-XaCS-NBmTyr(85)-P4	273
6	@157	391_F_CBmTyr(85)_P3	143_R_3'LET	390_LET_CLS-XaCS-P3-CBmTyr(85)	663
7	@157	142_F_5'LET	402_R_NBmTyr(157)_P4	400_LET_CLS-XaCS-NBmTyr(157)-P4	489
8	@157	411_F_CBmTyr(157)_P3	143_R_3'LET	410_LET_CLS-XaCS-P3-CBmTyr(157)	447
9	@157	142_F_5'LET	422_R_NBmTyr(201)_P4	420_LET_CLS-XaCS-NBmTyr(201)-P4	621
10	@157	431_F_CBmTyr(201)_P3	143_R_3'LET	430_LET_CLS-XaCS-P3-CBmTyr(201)	315

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), 2.5% DMSO and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA	10 ng	1 μL
DMSO	2.5 % (v/v)	0.5 μL
Nuclease-free water	–	6.5 μL

The reaction mixture was subjected to thermal cycling, which included 30 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	2 min	1
Denaturation	98°C	15 sec	30
Annealing	60°C	10 sec
Extension	72°C	45
Final extension	72°C	10 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

PCR (Platinum™ SuperFi™ II) with @156, @157, @162 and @166 for constructs 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201), 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M), 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M), LET-P3, and P4-LET

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from @156, @177, @162, and @166 for constructs 380_LET_CLS-XaCS-NBmTyr(85)-P4, 390_LET_CLS-XaCS-P3-CBmTyr(85), 400_LET_CLS-XaCS-NBmTyr(157)-P4, 410_LET_CLS-XaCS-P3-CBmTyr(157), 420_LET_CLS-XaCS-NBmTyr(201)-P4, 430_LET_CLS-XaCS-P3-CBmTyr(201), 450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M), 500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M), LET-P3, and LET-P4 with high fidelity and efficiency.

Protocol:

Nr. during cloning process	Template	Forward primer	Reverse primer	Cloning intermediate	Expected length	Extension time
1	@162	142_F_5'LET	452_R_HcTyr1(1-296)-TEVCS(M)_3'LET	450_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)	1,353	1 min
2	@166	142_F_5'LET	502_R_VsTyr(37-357)-TEVCS(M)_3'LET	500_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)	1,500	1 min
3	@156	144_F_3'LET	392_R_5'LET_P3	390_LET_CLS-XaCS-P3-CBmTyr(85), 410_LET_CLS-XaCS-P3-CBmTyr(157), 430_LET_CLS-XaCS-P3-CBmTyr(201)	940	45 sec
4	@156	381_F_3'LET_P4	145_R_5'LET	380_LET_CLS-XaCS-NBmTyr(85)-P4, 400_LET_CLS-XaCS-NBmTyr(157)-P4, 420_LET_CLS-XaCS-NBmTyr(201)-P4	940	45 sec
5	@157	142_F_5'LET	382_R_NBmTyr(85)_P4	380_LET_CLS-XaCS-NBmTyr(85)-P4	273	30 sec
6	@157	391_F_CBmTyr(85)_P3	143_R_3'LET	390_LET_CLS-XaCS-P3-CBmTyr(85)	663	45 sec
7	@157	142_F_5'LET	402_R_NBmTyr(157)_P4	400_LET_CLS-XaCS-NBmTyr(157)-P4	489	30 sec
8	@157	411_F_CBmTyr(157)_P3	143_R_3'LET	410_LET_CLS-XaCS-P3-CBmTyr(157)	447	30 sec
9	@157	142_F_5'LET	422_R_NBmTyr(201)_P4	420_LET_CLS-XaCS-NBmTyr(201)-P4	621	45 sec
10	@157	431_F_CBmTyr(201)_P3	143_R_3'LET	430_LET_CLS-XaCS-P3-CBmTyr(201)	315	30 sec

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA	10 ng	1 μL
DMSO	2.5 % (v/v)	0.5 µL
Nuclease-free water	–	6.5 μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98 °C	30 sec	1
Denaturation	98 °C	10 sec	35
Annealing	58 °C	20 sec
Extension	72 °C	130 seconds/kb
Final extension	72 °C	5 min	1
Hold	4 °C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

PCR (Platinum™ SuperFi™ II) for 330_LET_CLS-XaCS-BmTyr, 340_LET_CLS-XaCS-BmTyr(E195S+A221V), 350_LET_CLS-XaCS-BmTyr(R209S+M215E+V218M), 360_LET_CLS-XaCS-BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C), 370_LET_CLS-XaCS-cpBmTyr(201), 440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466), 460_LET_CLS-XaCS-LsTyr, 470_LET_CLS-XaCS-SkMelC1(33-124), 480_LET_CLS-XaCS-SkMelC2, 490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518), 510_LET_CLS-XaCS-SavMelC1(27-118), 520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F), 530_LET_CLS-XaCS-SavMelC2, and 540_LET_CLS-XaCS-SavMelC2(I42Y)

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from GA1-14 for constructs 330_LET_CLS-XaCS-BmTyr, 340_LET_CLS-XaCS-BmTyr(E195S+A221V), 350_LET_CLS-XaCS-BmTyr(R209S+M215E+V218M), 360_LET_CLS-XaCS-BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C), 370_LET_CLS-XaCS-cpBmTyr(201), 440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466), 460_LET_CLS-XaCS-LsTyr, 470_LET_CLS-XaCS-SkMelC1(33-124), 480_LET_CLS-XaCS-SkMelC2, 490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518), 510_LET_CLS-XaCS-SavMelC1(27-118), 520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F), 530_LET_CLS-XaCS-SavMelC2, and 540_LET_CLS-XaCS-SavMelC2(I42Y) with high fidelity and efficiency.

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	10-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	5 μL
Forward + Reverse Primers (pre-mixed at 5 μM each, 146-147)	0.5 μM	1 μL
Template DNA	10 ng	1 μL
Nuclease-free water	–	3 μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	10 sec	35
Annealing	60°C	10 sec
Extension	72°C	1min 20sec
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

Gibson Assembly for 330_LET_CLS-XaCS-BmTyr, 340_LET_CLS-XaCS-BmTyr(E195S+A221V), 350_LET_CLS-XaCS-BmTyr(R209S+M215E+V218M), 360_LET_CLS-XaCS-BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C), 370_LET_CLS-XaCS-cpBmTyr(201), 440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466), 460_LET_CLS-XaCS-LsTyr, 470_LET_CLS-XaCS-SkMelC1(33-124), 480_LET_CLS-XaCS-SkMelC2, 490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518), 510_LET_CLS-XaCS-SavMelC1(27-118), 520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F), 530_LET_CLS-XaCS-SavMelC2, and 540_LET_CLS-XaCS-SavMelC2(I42Y)

Goal:

Gibson assembly was performed to assemble the @156 vector and @157, @158, @159, @160, @161, @162, @163, @164, @165, @166, @167, @168, @169, @170 inserts to produce 330_LET_CLS-XaCS-BmTyr, 340_LET_CLS-XaCS-BmTyr(E195S+A221V), 350_LET_CLS-XaCS-BmTyr(R209S+M215E+V218M), 360_LET_CLS-XaCS-BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C), 370_LET_CLS-XaCS-cpBmTyr(201), 440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466), 460_LET_CLS-XaCS-LsTyr, 470_LET_CLS-XaCS-SkMelC1(33-124), 480_LET_CLS-XaCS-SkMelC2, 490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518), 510_LET_CLS-XaCS-SavMelC1(27-118), 520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F), 530_LET_CLS-XaCS-SavMelC2, and 540_LET_CLS-XaCS-SavMelC2(I42Y).

Protocol:

Nr. during cloning process	Vector	Insert	Construct
GA1	@156	@157	330_LET_CLS-XaCS-BmTyr
GA2	@156	@158	340_LET_CLS-XaCS-BmTyr(E195S+A221V)
GA3	@156	@159	350_LET_CLS-XaCS-BmTyr(R209S+M215E+V218M)
GA4	@156	@160	360_LET_CLS-XaCS-BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C)
GA5	@156	@161	370_LET_CLS-XaCS-cpBmTyr(201)
GA6	@156	@162	440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466)
GA7	@156	@163	460_LET_CLS-XaCS-LsTyr
GA8	@156	@164	470_LET_CLS-XaCS-SkMelC1(33-124)
GA9	@156	@165	480_LET_CLS-XaCS-SkMelC2
GA10	@156	@166	490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518)
GA11	@156	@167	510_LET_CLS-XaCS-SavMelC1(27-118)
GA12	@156	@168	520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F)
GA13	@156	@169	530_LET_CLS-XaCS-SavMelC2
GA14	@156	@170	540_LET_CLS-XaCS-SavMelC2(I42Y)

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Components
NEBuilder HiFi DNA Assembly Master Mix	10 µL
Vector	20 ng = 2 µL
Insert	20 ng = 2 µL
Nuclease-free Water	6 μL
Total	20 µL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of Gibson assembly was evaluated by downstream experiments.

Agarose Gel Electrophoresis for 330_LET_CLS-XaCS-BmTyr, 340_LET_CLS-XaCS-BmTyr(E195S+A221V), 350_LET_CLS-XaCS-BmTyr(R209S+M215E+V218M), 360_LET_CLS-XaCS-BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C), 370_LET_CLS-XaCS-cpBmTyr(201), 440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466), 460_LET_CLS-XaCS-LsTyr, 470_LET_CLS-XaCS-SkMelC1(33-124), 480_LET_CLS-XaCS-SkMelC2, 490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518), 510_LET_CLS-XaCS-SavMelC1(27-118), 520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F), 530_LET_CLS-XaCS-SavMelC2, and 540_LET_CLS-XaCS-SavMelC2(I42Y)

Goal:

An analytical agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples (330_LET_CLS-XaCS-BmTyr, 340_LET_CLS-XaCS-BmTyr(E195S+A221V), 350_LET_CLS-XaCS-BmTyr(R209S+M215E+V218M), 360_LET_CLS-XaCS-BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C), 370_LET_CLS-XaCS-cpBmTyr(201), 440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466), 460_LET_CLS-XaCS-LsTyr, 470_LET_CLS-XaCS-SkMelC1(33-124), 480_LET_CLS-XaCS-SkMelC2, 490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518), 510_LET_CLS-XaCS-SavMelC1(27-118), 520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F), 530_LET_CLS-XaCS-SavMelC2, and 540_LET_CLS-XaCS-SavMelC2(I42Y)) based on their size.

Protocol:

Agarose gel electrophoresis was performed using a 2% w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right:

Ladder - 330_LET_CLS-XaCS-BmTyr - 340_LET_CLS-XaCS-BmTyr(E195S+A221V) - 350_LET_CLS-XaCS-BmTyr(R209S+M215E+V218M) - 360_LET_CLS-XaCS-BmTyr(G43R+M61H+F197W+Q214D+V217A+A232C) - 370_LET_CLS-XaCS-cpBmTyr(201) - 440_LET_CLS-XaCS-HcTyr1(1-296)-TEVCS(M)-HcTyr1(336-466) - 460_LET_CLS-XaCS-LsTyr - 470_LET_CLS-XaCS-SkMelC1(33-124) - 480_LET_CLS-XaCS-SkMelC2 - 490_LET_CLS-XaCS-VsTyr(37-357)-TEVCS(M)-VsTyr(371-518) - 510_LET_CLS-XaCS-SavMelC1(27-118) - 520_LET_CLS-XaCS-SavMelC1(27-118)(Y92F) - 530_LET_CLS-XaCS-SavMelC2 - 540_LET_CLS-XaCS-SavMelC2(I42Y)

gBlocks resuspension: @156, @157, @162 and @166

Goal:

The gBlocks @156, @157, @162 and @166 from TWIST Bioscience were resuspended in IDTE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) to a final concentration of 10 ng/μL.

Protocol:

Lyophilized gBlocks were briefly centrifuged to collect the pellet at the bottom of the tube. The DNA was resuspended by vortexing in nuclease-free IDTE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) to reach the desired concentration of 10 ng/μL.

Results:

The resuspended gBlocks were stored at −20 °C and used for subsequent downstream experiments.

PCR (Platinum™ SuperFi™ II) for 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K), and 320_pcDNAT7v2

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 200_pcDNAT7v1, 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, and 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) for constructs 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K), and 320_pcDNAT7v2, respectively, with high fidelity and efficiency.

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA	10 ng	1 μL
DMSO	2.5% (v/v)	0.5 μL
Nuclease-free water	–	6.5 μL

As reaction templates, yet unverified clones were used, with the reaction label differentiated by the clone number of the template in question. Components of individual reactions are defined as follows:

No.	Template	Forward primer	Reverse Primer	Amplicon size	Extension time	Cloning intermediate
31	210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E)	221_F_NTEVp(E75S)	222_R_NTEVp(E75S)	7042 bp	4:00 min	220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S)
32	250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP	231_F_Stop-NcoI-T7tt	232_R_CTEVp	7042 bp	4:00 min	230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)
33	250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP	241_F_BFP	242_R_s(GS)L	7804 bp	4:30 min	240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP
34	250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP	271_F_tTA-(G4S)2L	272_R_TetR	8455 bp	5:00 min	270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP
35	290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)	221_F_NTEVp(E75S)	222_R_NTEVp(E75S)	7549 bp	4:30 min	300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K)
36	200_pcDNAT7v1	321_F_T7tt_HindIII	322_R_T7p_KpnI	6051 bp	3:30 min	320_pcDNAT7v2

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 30 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	2 min	1
Denaturation	98°C	15 sec	35
Annealing	62°C	10 sec
Extension	72°C	30 seconds/kb
Final extension	72°C	10 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

Plasmid Maxiprep (QIAGEN) of 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 320_pcDNAT7v2, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) and 550_pCas9_sgAAVS1_Cas9-HA-2xNLS-i53

Goal:

A maxiprep of 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 320_pcDNAT7v2, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) and 550_pCas9_sgAAVS1_Cas9-HA-2xNLS-i53 was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Protocol:

DNA plasmids were prepared using the QIAGEN ® Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). 200 mL of overnight culture of E. coli was centrifuged at 4500 × g for 20 min at 4°C. The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer with thorough mixing. The mixture was centrifuged at 7000 × g (eppendorf 5430 R) for 135 min (if supernatant not clear, optional +30) min at 4 °C. The supernatant was applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuging at 7000 × g for 120 min (if pellet not strong enough, additional 30 min) at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol. Finally, the supernatant was aspirated using a vacuum pump without disturbing the pellet, and the pellet was air-dried for 5–10 min. The DNA was then redissolved in 200 μL of TE buffer, the concentration was determined using a NanoDrop, and the DNA was stored at −20 °C.

Results:

The maxiprep procedure yielded DNA of sufficient quantity (apart from sample 290 which had very low yield) and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
210	1282	1.95	2.25
220	3200	1.92	2.2
250	2186	1.94	2.24
290	182	1.91	2.27
320	565	1.91	2.22
550	598	1.94	2.23

E. coli transformation 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

Transformation was performed to introduce plasmid DNA constructs 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 into DH10β E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

For each construct, an aliquot of competent DH10β E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells (1–5 μL) and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 μL of room temperature SOC, the mixture was then recovered for 30–60 min at 37 °C with shaking (#Heatblock). The mixture was centrifuged at 16000 x g for 30 seconds to remove the supernatant. The pellet was resuspended in 50 μl of LB and spread onto a selection plate and incubated overnight at 37°C.

Results:

Agarose Gel Electrophoresis 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP 280_pcDNAT7v1_RS20-cpEFGP-CaM 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Protocol:

Agarose gel electrophoresis was performed using a 1% w/v agarose gel (Carl ROTH) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5–10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right: …. Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

KLD of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP

Goal:

KLD reaction was performed to assemble construct 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP from PCR products 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP respectively.

Protocol:

KLD reaction was performed using the KLD Enzyme Mix (M0554, New England Biolabs), consisting of a kinase, a ligase, and a DpnI restriction enzyme, according to the manufacturer's instructions. For each reaction, KLD reaction buffer, KLD enzyme mix, nuclease-free water, and the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 μL
KLD Reaction Buffer (2x)	5 μL
KLD Enzyme Mix (10x)	1 μL
Nuclease-free water	3 μL
Total	10 μL

The sample was incubated at room temperature (25 °C) for 5–10 minutes. Optionally, to increase the efficiency of the DpnI digestion, the mixture was additionally incubated at 37 °C for 30–60 minutes. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of KLD reaction was evaluated by downstream experiments.

Gibson Assembly of 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 v2 & v3

Goal:

Gibson assembly was performed to assemble the ... vector and ... inserts to produce ...

Protocol:

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Components	280_pcDNAT7v1_RS20-cpEFGP-CaM H(olger)	280_pcDNAT7v1_RS20-cpEFGP-CaM L(uitgard)	290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) I(rmgard)	290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) M(echthild)	310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 J(utta)	310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 N(orbert)
NEBuilder HiFi DNA Assembly Master Mix	10 μL	10 μL	10 μL	10 μL	10 μL	10 μL
Backbone	1.7 μl (0.050 pmol)	1.7 μl (0.050 pmol)	1.3 μl (0.050 pmol)	1.3 μl (0.050 pmol)	1.2 μl (0.050 pmol)	1.2 μl (0.050 pmol)
Insert	1.3 μl (0.100 pmol)	0.4 μl (0.100 pmol)	0.9 μl (0.100 pmol)	0.4 μl (0.100 pmol)	0.8 μl (0.100 pmol)	0.5 μl (0.100 pmol)
Nuclease-free Water	7 μl	7.9 μl	7.8 μl	8.3 μl	7.9 μl	8.3 μl
Total	20 μL	20 μL	20 μL	20 μL	20 μL	20 μL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of Gibson assembly was evaluated by downstream experiments.

Gibson Assembly of 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

Gibson assembly was performed to assemble the 200_pcDNAT7v1 vector and @153, @154, @155 inserts to produce 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4.

Protocol:

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Components	Volume
NEBuilder HiFi DNA Assembly Master Mix	10 µL
Vector	3.5 / 2.6 / 2.4 μL
Insert	0.7 / 0.5 / 0.3 μL
Nuclease-free Water	... μL
Total	20 µL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of Gibson assembly was evaluated by downstream experiments.

Plasmid Maxiprep (QIAGEN) of 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, and 560_pAAVS1_CAGp-IL7Ra-IRES-BSD

Goal:

A maxiprep of 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, and 560_pAAVS1_CAGp-IL7Ra-IRES-BSD (and 560_pAAVS1_CAGp-IL7Ra-IRES-BSD re-inoculated) was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Protocol:

DNA plasmids were prepared using the QIAGEN ® Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). 200 mL of overnight culture of E. coli was centrifuged at 4500 × g for 20 min at 4°C. The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer with thorough mixing. The mixture was centrifuged at 7000 × g (eppendorf 5430 R) for 135 min (if supernatant not clear, optional +30) min at 4 °C. The supernatant was applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuging at 7000 × g for 120 min (if pellet not strong enough, additional 30 min) at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol. Finally, the supernatant was aspirated using a vacuum pump without disturbing the pellet, and the pellet was air-dried for 5–10 min. The DNA was then redissolved in 200 μL of TE buffer, the concentration was determined using a NanoDrop, and the DNA was stored at −20 °C.

Results:

The maxiprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
230	1728	1.93	2.25
240	1832	1.95	2.17
560 re-inoc.	2990	1.95	2.21
560	1215	1.95	2.26

Agarose gel electrophoresis of 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K) digest

Goal:

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Protocol:

Agarose gel electrophoresis was performed using a 1.0% w/v agarose gel (Carl ROTH) prepared in 1×TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 3 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1×TAE buffer and connected to a power supply. A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Geldoc_model).

Results:

The following samples were loaded into the gel from left to right: NEB 1 kb Plus DNA Ladder, 330.5–330.12 digest.  Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples for samples 330.6 and 330.7.

Sanger sequencing of 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal:

To confirm the nucleotide sequences of 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K), Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a (partial) match between the experimental and expected sequences. In addition, the sequencing data showed (predominantly) a high quality score and a continuous read length of over 1 kb.

Sample	Barcode	Primer	Validation
300.1	06817491	T7	Deletion in cloning region
300.1	06817492	T7-Term	Deletion in cloning region
300.2	06817493	T7	Deletion in cloning region
300.2	06817494	T7-Term	Deletion in cloning region
300.3	06817495	T7	Aligns with 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S)
300.3	06817496	T7-Term	Aligns with 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S)
300.4	06817497	T7	Aligns with 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S)
300.4	06817498	T7-Term	Aligns with 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S)

E. coli colony picking of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP

Goal

Colony picking of individual DH10β E. coli (Cat. No. C3019, New England Biolabs) bacterial colonies transformed with 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP from selection plates containing carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plates. Each colony was transferred to 5 mL of fresh culture medium (LB medium with 100 μg/mL carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm (CERTOMAT BS-1 Incubator Shaker, Sartorius) for further growth and amplification of the selected cells. The agar plates were sealed to prevent contamination and stored at 4 °C.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial cultures after overnight incubation at 37 °C.

E. coli transformation of 320_pcDNAT7v2, 550_pCas9_sgAAVS1_Cas9-HA-2xNLS-i53, and 560_pAAVS1_CAGp-IL7Ra-IRES-BSD

Goal:

Transformation was performed to introduce plasmid DNA constructs 320_pcDNAT7v2, 550_pCas9_sgAAVS1_Cas9-HA-2xNLS-i53, and 560_pAAVS1_CAGp-IL7Ra-IRES-BSD into DH10β E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

For each construct, an aliquot of competent DH10β E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells (1–5 μL) and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 μL of room temperature SOC, the mixture was then recovered for 30–60 min at 37 °C with shaking (#Heatblock). 50 μl of the mixture was spread onto a selection plate and incubated overnight at 37°C. The remaining volume of 150 μl was used for inoculation of a 200 mL culture (LB medium with 100 μg/mL carbenicillin), which was incubated overnight at 37 °C with continuous shaking at 200 rpm (CERTOMAT BS-1 Incubator Shaker, Sartorius).

Results:

Plasmid Miniprep (NEB) of 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal:

Minipreps of 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K) were performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol:

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight cultures were pelleted by centrifugation for 15 minutes at 4000 ×  g (Eppendorf, Centrifuge 5804R). For each sample, the supernatant was discarded. Pellet was resuspended in 200 μL of plasmid resuspension buffer. Cell lysis was achieved by the addition of 200 μL of plasmid lysis buffer, followed by 5–6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of plasmid neutralization buffer. The tubes were gently inverted and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transferred to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of plasmid wash buffer 1 and centrifuging for 1 minute, followed by 400 μL of plasmid wash buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL Eppendorf tubes. DNA was eluted by the addition of 30 μL of preheated TE buffer (55–65 °C) to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop (#Nano_model), and the DNA samples were stored at −20 °C.

Results:

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
300.1	768.30	1.95	2.38
300.2	761.33	1.94	2.35
300.3	688.87	1.93	2.38
300.4	703.48	1.94	2.35
300.5	717.11	1.94	2.35
300.6	749.18	1.95	2.34
300.7	662.50	1.94	2.33
300.8	700.76	1.93	2.33
300.9	688.44	1.92	2.26
300.10	694.97	1.93	2.32
300.11	745.75	1.93	2.30
300.12	698.68	1.92	2.31

Sanger sequencing of KLD 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP and 320_pcDNAT7v2

Goal:

To confirm the nucleotide sequences of 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP and 320_pcDNAT7v2, Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a (partial) match between the experimental and expected sequences. In addition, the sequencing data showed (predominantly) a high quality score and a continuous read length of over 1 kb.

Sample	Barcode	Primer	Validation
270a.1	06817487	T7	No priming
270a.1	06817488	T7-Term	No priming
320.3	06817489	CMV-Forward	Success
320.4	06817490	CMV-Forward	Multiple point mutations outside of the cloning region

E. coli transformation of 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, and 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal:

Transformation was performed to introduce plasmid DNA constructs 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, and 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K) into DH10β E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

For each construct, an aliquot of competent DH10β E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells (1–5 μL) and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 μL of room temperature SOC, the mixture was then recovered for 30–60 min at 37 °C with shaking (#Heatblock). 50 μl of the mixture was spread onto a selection plate and incubated overnight at 37°C. The remaining volume of 150 μl was used for inoculation of a 200 mL culture (LB medium with 100 μg/mL carbenicillin), which was incubated overnight at 37 °C with continuous shaking at 200 rpm (CERTOMAT BS-1 Incubator Shaker, Sartorius).

Results:

Plasmid Miniprep (NEB) of KLD 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K) and 320_pcDNAT7v2

Goal:

Minipreps of 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K) and 320_pcDNAT7v2 were performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol:

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight cultures were pelleted by centrifugation for 15 minutes at 4000 ×  g (Eppendorf, Centrifuge 5804R). For each sample, the supernatant was discarded. Pellet was resuspended in 200 μL of plasmid resuspension buffer. Cell lysis was achieved by the addition of 200 μL of plasmid lysis buffer, followed by 5–6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of plasmid neutralization buffer. The tubes were gently inverted and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transferred to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of plasmid wash buffer 1 and centrifuging for 1 minute, followed by 400 μL of plasmid wash buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL Eppendorf tubes. DNA was eluted by the addition of 30 μL of preheated TE buffer (55–65 °C) to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop (#Nano_model), and the DNA samples were stored at −20 °C.

Results:

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
220a.1	30.5	1.9	1.9
220a.2	56.9	1.9	2.0
220a.3	72.4	1.9	1.8
220a.4	36.6	1.9	1.6
230a.1	70.4	1.9	1.8
230a.2	95.6	1.9	1.7
230b.1	30.6	1.7	1.0
230b.2	32.8	1.9	1.6
240a.1	78.2	1.9	1.8
240a.2	67.3	1.9	1.9
240b.1	35.7	1.7	1.4
240b.2	12.3	1.5	6.7
270a.1	42.6	1.9	1.8
270a.2	98.2	2.0	1.9
270b.1	37.8	1.9	1.8
270b.2	10.7	1.4	0.5
300a.1	94.1	1.9	2.1
300a.2	127.8	2.1	2.0
300b.1	27.2	1.8	1.5
300b.2	61.4	2.0	1.9
320.1	21.6	1.5	0.6
320.2	60.5	1.5	1.6
320.3	30.8	2.0	1.0
320.4	85.7	1.9	2.0

E. coli colony picking of 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K)

Goal

Colony picking of individual DH10β E. coli (Cat. No. C3019, New England Biolabs) bacterial colonies transformed with 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K) from selection plates containing carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plates. Each colony was transferred to 5 mL of fresh culture medium (LB medium with 100 μg/mL carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm (CERTOMAT BS-1 Incubator Shaker, Sartorius) for further growth and amplification of the selected cells. The agar plates were sealed to prevent contamination and stored at 4 °C.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial cultures after overnight incubation at 37 °C.

KLD of 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K), and 320_pcDNAT7v2

Goal:

KLD reaction was performed to assemble constructs 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K), and 320_pcDNAT7v2 from PCR products 31.1, 32.1, 32.2, 33.1, 33.2, 34.1, 34.1, 35.1, 35.2, and 36, respectively.

Protocol:

KLD reaction was performed using the KLD Enzyme Mix (M0554, New England Biolabs), consisting of a kinase, a ligase, and a DpnI restriction enzyme, according to the manufacturer's instructions. For each reaction, KLD reaction buffer, KLD enzyme mix, nuclease-free water, and the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 μL
KLD Reaction Buffer (2x)	5 μL
KLD Enzyme Mix (10x)	1 μL
Nuclease-free water	3 μL
Total	10 μL

The sample was incubated at room temperature (25 °C) for 5–10 minutes. To increase the efficiency of the DpnI digestion, the mixture was additionally incubated at 37 °C for 30–60 minutes. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of KLD reaction was evaluated by downstream experiments.

Sanger sequencing of KLD 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K) and 320_pcDNAT7v2

Goal:

To confirm the nucleotide sequences of 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K), and 320_pcDNAT7v2, Sanger sequencing was performed using GENEWIZ, Azenta services.

Protocol:

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) following the sample submission guidelines. For each sample, 10 μL of purified plasmid at a concentration of 50–100 ng/μL or 10 μL of purified PCR fragments at a concentration of 2–6 ng/μL were added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results:

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious Prime. The alignment analysis showed a (partial) match between the experimental and expected sequences. In addition, the sequencing data showed (predominantly) a high quality score and a continuous read length of over 1 kb.

Sample	Barcode	Primer	Validation
220a.3	6817458	T7	Success
220a.3	6817459	T7-Term	Success
230a.1	6817460	T7	Success
230a.1	6817461	T7-Term	Success
230a.2	6817462	T7	Success
230a.2	6817463	T7-Term	Success
230b.1	6817464	T7	Success
230b.1	6817465	T7-Term	Success
230b.2	6817466	T7	Success
230b.2	6817467	T7-Term	Success
240a.1	6817468	T7	Deletion at the cloning site
240a.1	6817469	T7-Term	Deletion at the cloning site
240a.2	6817470	T7	Success
240a.2	6817471	T7-Term	Success
240b.1	6817472	T7	Success
240b.1	6817473	T7-Term	Success
270a.2	6817474	T7	Partially missing elements of CDS
270a.2	6817475	T7-Term	Partially missing elements of CDS
270b.1	6817476	T7	Deletion at the cloning site
270b.1	6817477	T7-Term	Deletion at the cloning site
300a.1	6817478	T7	Missing insert
300a.1	6817479	T7-Term	Missing insert
300a.2	6817480	T7	Align with 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S)
300a.2	6817481	T7-Term	Align with 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S)
300b.1	6817482	T7	Align with 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S)
300b.1	6817483	T7-Term	Align with 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S)
300b.2	6817484	T7	Align with 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S)
300b.2	6817485	T7-Term	Align with 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S)
320.2	6817486	CMV-Forward	Deletion at the cloning site

PCR (Platinum™ SuperFi™ II) for insert amplification from @150–@155

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from @150, @151, @152, @153, @154, and @155 for constructs 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4, respectively, with high fidelity and efficiency.

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA	10 ng	1 μL
Nuclease-free water	–	7 μL

Components of individual reactions are defined as follows:

No.	Template	Forward primer	Reverse Primer	Amplicon size	Extension time	Cloning intermediate
25	@150	251_F_SacI-Kozak-CD4SP_T7p	212_R_NTEVp-Stop-NcoI_T7tt	1015 bp	30 s	210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E)
26	@151	251_F_SacI-Kozak-CD4SP_T7p	252_R_BFP-Stop-NcoI_T7tt	2807 bp	90 s	250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP
27	@152	251_F_SacI-Kozak-CD4SP_T7p	252_R_BFP-Stop-NcoI_T7tt	2549 bp	90 s	260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP
28	@153	281_F_SacI-RS20_T7p	282_R_CaM-NcoI_pcDNA	1287 bp	45 s	280_pcDNAT7v1_RS20-cpEFGP-CaM
29	@154	281_F_SacI-RS20_T7p	292_R_CTEVp-Stop-NcoI_T7tt	1525 bp	45 s	290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)
30	@155	281_F_SacI-RS20_T7p	312_R_P4-Stop-NcoI_T7tt	688 bp	30 s	310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 30 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	2 min	1
Denaturation	98°C	15 sec	30
Annealing	60°C	10 sec
Extension	72°C	30 seconds/kb
Final extension	72°C	10 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

DNA dephosphorylation of digested 200_pcDNAT7v1

Goal:

Dephosphorylation of 5′ ends of DNA of digested 200_pcDNAT7v1 was performed for to prevent re-ligation of the digested vector.

Protocol:

Dephosphorylation of 5′ ends of DNA was carried out using Quick CIP ( ...,   New England Biolabs) according to the manufacturers protocol. Template DNA was supplied in amount of x pmol of DNA ends (about 2.5 μg of a 6 kB plasmid) combined with 5 μL of Quick CIP in a PCR-tube. The sample was mixed by pipetting, and incubated at 37°C for 10 mins. The reaction was then stopped using heat inactivation in 80°C for 20 minutes.

The reaction was stored at -20°C or immediately used for downstream experiments .

Results:

The efficiency of dephosphorylation of DNA ends was evaluated by downstream experiments.

PCR (Platinum™ SuperFi™ II): 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from gBlocks @150, @151, @152, @153, @154, and 155 for constructs 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 with high fidelity and efficiency.

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	1 μL
Template DNA	50 ng	5 μL
Nuclease-free water	–	4 μL

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 sec	1
Denaturation	98°C	5–10 sec	35
Annealing	60°C	10 sec
Extension	72°C	35 sec (12, 17) 50 sec (16) 1 min 20 sec (14, 15) 1 min 35 sec (13)
Final extension	72°C	5 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

E. coli transformation with 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

Transformation was performed to introduce plasmid DNA constructs 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 into DH10β E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

An aliquot of competent DH10β E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells (7 μL) and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 45 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 μL of room temperature SOC, 50–100 μl of the mixture was spread onto a selection plate and incubated overnight at 37°C. If a selection other than ampicillin was used, the mixture was first recovered at 37 °C with shaking (#Heatblock) prior to plating.

Results:

E. coli colony picking for 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K), and 320_pcDNAT7v2

Goal

Colony picking of individual DH10β E. coli (Cat. No. C3019, New England Biolabs) bacterial colonies transformed with 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K), and 320_pcDNAT7v2 from selection plates containing carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plates. Each colony was transferred to 5 mL of fresh culture medium (LB medium with 100 μg/mL carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm (CERTOMAT BS-1 Incubator Shaker, Sartorius) for further growth and amplification of the selected cells. The agar plates were sealed to prevent contamination and stored at 4 °C.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial cultures after overnight incubation at 37 °C.

E. coli transformation with KLD 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K), and 320_pcDNAT7v2

Goal:

Transformation was performed to introduce plasmid DNA constructs 220_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75S), 230_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K), 240_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-BFP, 270_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-TetR-(G4S)2-BFP, 300_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75S)-T2A-FRB-(G4S)3-CTEVp(190K), and 320_pcDNAT7v2 into DH10β E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

For each construct, an aliquot of competent DH10β E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells (1–5 μL) and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 200 μL of room temperature SOC, the mixture was then recovered for 30–60 min at 37 °C with shaking (#Heatblock). The mixture was centrifuged at 16000 x g for 30 seconds to remove the supernatant. The pellet was resuspended in 50 μl of LB and spread onto a selection plate and incubated overnight at 37°C.

Results:

E. coli transformation with 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

Transformation was performed to introduce plasmid DNA constructs 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 into DH10β E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

For each construct, an aliquot of competent DH10β E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells (7 μL) and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following addition of 300 μL of room temperature LB, the mixtures were recovered for 30 minutes at 37°C with shaking (#Heatblock). The mixture was centrifuged at 16000 x g for 30 seconds to remove the supernatant. The pellet was resuspended in 50 μl of LB and spread onto a selection plate and incubated overnight at 37°C.

Results:

Successful transformation was confirmed by the presence of colonies on the selective agar plate.

E. coli colony picking of 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

Colony picking of individual DH10β E. coli bacterial colonies transformed with @20, 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 from selection plates containing carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol:

Using a sterile inoculationpipette tip, individual colonies were carefully picked from the surface of the selection plates. Each colony was transferred to 5 mL of fresh culture medium (LB medium with 100 µg/mL carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plates for GA310 & GA280 were sealed to prevent contamination and transferred to a fridge.

Construct	Sample
250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP	GA250.1
	GA250.2
260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP	GA260
280_pcDNAT7v1_RS20-cpEFGP-CaM	GA280.1
	GA280.2
290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)	GA290
310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4	GA310.1
	GA310.2

Results:

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation at 37°C.

Gibson assembly of 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

Gibson assembly was performed to assemble the backbone (samples 20–24) and insert (samples 25–30) amplicons to produce 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 constructs.

Protocol:

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including ca. 100 ng of vector with 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Components	210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E)	250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP	260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP	280_pcDNAT7v1_RS20-cpEFGP-CaM	290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)	310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4
NEBuilder HiFi DNA Assembly Master Mix	10 μL	10 μL	10 μL	10 μL	10 μL	10 μL
Backbone	2.0 μL	2.0 μL	2.0 μL	2.0 μL	2.0 μL	2.0 μL
Insert	1.8 μL	2.6 μL	2.3 μL	3.2 μL	2.4 μL	2.1 μL
Nuclease-free Water	6.2 μL	5.4 μL	5.7 μL	4.8 μL	5.6 μL	5.9 μL
Total	20 μL	20 μL	20 μL	20 μL	20 μL	20 μL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results:

The efficiency of Gibson assembly was evaluated by downstream experiments.

E. coli colony picking of 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

Colony picking of individual DH10β E. coli bacterial colonies transformed with 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 from selection plates containing carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol:

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plates. Each colony was transferred to 5 mL of fresh culture medium (LB medium with 100 μg/ml carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plates were sealed to prevent contamination and transferred to a fridge.

Results:

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Preparation of carbenicillin LB-Agar plates

Goal:

Preparation of Carbenicillin LB-Agar plates.

Protocol:

A bottle containing sterile LB-Agar was heated in the microwave until fully liquefied. After cooling to hand-warm temperature, the carbenicillin antibiotic was added 1:1000, i.e. from its stock of 100 mg/μl to the final concentration of 100 μg/μl. Plates were poured under the laminar flow hood and allowed to solidify (~15 mins).

Results:

Plates were successfully prepared and stored in the cold room for downstream applications.

Preparation of carbenicillin LB-Agar plates

Goal:

Preparation of Carbenicillin LB-Agar plates.

Protocol:

A bottle containing sterile LB-Agar was heated in the microwave until fully liquefied. After cooling to hand-warm temperature, the carbenicillin antibiotic was added 1:1000, i.e. from its stock of 100 mg/μl to the final concentration of 100 μg/μl. Plates were poured under the laminar flow hood and allowed to solidify (~15 mins).

Results:

Plates were successfully prepared and stored in the cold room for downstream applications.

DpnI digest of backbone amplicons from 200_pcDNAT7v1

Goal:

Restriction digest of the backbone PCR fragments of 200_pcDNAT7v1 (samples 20–24) with DpnI enzyme, for downstream cloning of 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4.

Protocol:

Restriction digest was carried out using the DpnI enzyme (R0176, New England Biolabs)  according to the manufacturer's instructions. Template DNA was supplied in an amount of 1.5 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of DpnI in a 1.5 mL microcentrifuge tube. The sample was mixed by pipetting and incubated at 37°C for 30 min.

Component	Volume
Restriction enzyme	10 U (1 μL)
rCutSmart buffer	2.5 μL
DNA	1.5 μg (19 μL)
Nuclease-free Water	to 25 μL

The reaction was then stopped using heat inactivation (80°C, 10 minutes). The product was stored at -20°C or immediately used for downstream experiments.

Results:

The efficiency of the restriction digest was evaluated by downstream experiments.

E. coli transformation with 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

Transformation was performed to introduce plasmid DNA constructs 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol:

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10–100 ng/μL, was added to the cells (1–5 μL) and gently mixed by flicking the tube 4–5 times. The cell-DNA mixture was incubated on ice for 10 minutes. Following the incubation period, heatshock at 42°C for 30 seconds (#Heatblock) was performed. Subsequently, the cells were placed on ice for 3 minutes. Following the addition of 200 μL of room temperature SOC, 50–100 μl of the mixture was spread onto a selection plate and incubated overnight at 37°C. If a selection other than ampicillin was used, the mixture was first recovered at 37 °C with shaking (#Heatblock) prior to plating.

Results:

PCR Clean-up (NEB) of fragments for 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications.

Protocol:

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to the manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:5 for dsDNA and mixed by pipetting up and down. The suspension was loaded onto the columns and centrifuged for 1 minute at 13000 × g (TableTop_model).  The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g, followed by 30 sec centrifugation at 13000 x g for drying. The columns were transferred to clean 1.5 mL minicentrifuge tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by the addition of 10 μL of preheated TE buffer (55–65 °C) to the column. Following 5 minute of incubation, the samples were centrifuged for 1 minute at 13000 × g. DNA concentration was determined using a NanoDrop (Nano_model), and the DNA samples were stored at −20 °C.

Results:

The PCR Clean-up yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
20	74.46	1.95	4.62
21	51.25	1.95	7.15
22	64.99	1.90	4.47
23	78.20	1.95	3.95
24	73.88	1.91	4.20
25	52.17	1.96	5.76
26	69.27	1.92	3.89
27	72.12	1.91	4.70
28	32.75	1.93	-45.51
29	61.91	1.94	5.79
30	67.47	1.89	5.36

PCR (Platinum™ SuperFi™ II) for backbone amplification from 200_pcDNAT7v1

Goal:

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 200_pcDNAT7v1 for constructs 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4, with high fidelity and efficiency.

Protocol:

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 10 pmol of each forward and reverse primers (final concentration 0.5 μM), and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop (Nano_model). The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each)	0.5 μM	2 μL
Template DNA	10 ng	1 μL
Nuclease-free water	–	7 μL

Components of individual reactions are defined as follows:

No.	Template	Forward primer	Reverse Primer	Amplicon size	Extension time	Cloning intermediate
20	200_pcDNAT7v1	213_F_NcoI-T7tt_NTEVp-Stop	254_R_T7p-SacI_Kozak-CD4SP	6066 bp	210 s	210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E)
21	200_pcDNAT7v1	253_F_NcoI-T7tt_BFP-Stop	254_R_T7p-SacI_Kozak-CD4SP	6066 bp	210 s	250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP
22	200_pcDNAT7v1	283_F_NcoI-pcDNA_CaM	254_R_T7p-SacI_Kozak-CD4SP	6063 bp	210 s	280_pcDNAT7v1_RS20-cpEFGP-CaM
23	200_pcDNAT7v1	293_F_NcoI-T7tt_CTEVp-Stop	254_R_T7p-SacI_Kozak-CD4SP	6062 bp	210 s	290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K)
24	200_pcDNAT7v1	313_F_NcoI-T7tt_P4-Stop	254_R_T7p-SacI_Kozak-CD4SP	6062 bp	210 s	310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

The reaction mixture was subjected to thermal cycling (Cycler_model), which included 30 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	2 min	1
Denaturation	98°C	15 sec	30
Annealing	60°C	10 sec
Extension	72°C	30 seconds/kb
Final extension	72°C	10 min	1
Hold	4°C	∞	1

Amplified DNA products were kept at +4°C for short-term or -20°C for long-term storage until further use.

Results:

The efficiency of PCR was evaluated by downstream experiments.

Plasmid Miniprep (NEB) of 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4

Goal:

A miniprep of the liquid cultures containing 210_pcDNAT7v1_CD4SP-HA-LaM4-(G2S)6-CD28TMD-NTEVp(75E), 250_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-tTA-(G4S)2-BFP, 260_pcDNAT7v1_CD4SP-HA-LaM2-(G2S)6-CD28TMD-CTEVp(190K)-TEVCS(M)-rtTA-(G4S)2-BFP, 280_pcDNAT7v1_RS20-cpEFGP-CaM, 290_pcDNAT7v1_HA-FKBP-(G4S)3-NTEVp(75E)-T2A-FRB-(G4S)3-CTEVp(190K), and 310_pcDNAT7v1_Myc-P3h-CS(M)-CAD-CS(M)-hP4 was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol:

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 ×  g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop (#Nano_model).

Results:

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	A260/280	A260/230
210.1	108.90	1.76	2.29
210.2	63.86	1.95	3.33
210.3	70.73	1.86	2.78
250.1	129.39	1.94	2.55
250.2	49.09	1.94	3.12
250.3	89.18	1.94	2.68
260.1	129.31	1.94	2.61
260.2	111.41	1.95	2.57
260.3	95.94	1.91	2.60
280.1	88.66	1.92	2.75
280.2	120.96	1.92	2.72
280.3	50.90	1.87	3.71
290.1	158.40	1.91	2.49
290.2	86.56	1.92	2.83
290.3	81.58	1.91	2.99
310.1	146.53	1.92	2.54
310.2	36.73	1.91	7.67
310.3	72.59	1.85	2.80

Restriction digest

Goal:

Base

Restriction digest of the backbone 200.1 and 200.2 with SacI-HF & NcoI-HF enzyme, for downstream cloning of the constructs 210, 250, 260, 280, 290, and 310. (redo)

Protocol:

Restriction digest was carried out using the SacI-HF & NcoI-HFenzyme(s) ( ...,   New England Biolabs)  according to the manufacturers protocol. Template DNA was supplied in amount of 1 μg and combined with 5 μL of rCutSmart (B7204, New England Biolabs) and 1 μL of SacI-HF & NcoI-HF in a PCR tube. The sample was mixed by pipetting, and incubated at 37°C for 5 hour. The reaction was then stopped using heat inactivation (80°C, ... minutes).

Component	Volume
Restriction enzyme	10 U (1 μL)
rCutSmart buffer	5 μL
DNA	1 μg (0.5 µL)
Nuclease-free Water	To 50 μL

The reaction was then stored at -20°C or immediately used for downstream experiments .

Results:

The efficiency of restriction digest was evaluated by downstream experiments.

DNA phosphorylation of digested backbone 200

Goal:

Phosphorylation of 5′ ends of DNA of digested backbone 200 was performed for to enable downstream ligation.

Protocol:

Phosphorylation of 5′ ends of DNA was carried out using T4 PNK (M0201S, New England Biolabs) according to the manufacturers protocol. Template DNA was supplied in amount of up to 300 pmol of DNA ends and combined with 5 μL of T4 PNK Reaction Buffer (10X) (B0201S, New England Biolabs), 5 μL of 10mM ATP and 1 μL (10 units) of T4 PNK in a PCR-tube. The sample was mixed by pipetting, and incubated at 37°C for 30 mins. The reaction was then stopped using heat inactivation in 65°C for 20 minutes.

Component	Volume
DNA	Up to 300 pmol of 5' DNA ends
T4 PNK Reaction Buffer (10x)	5 μL
ATP (10mM)	5 μL
T4 PNK	1 μL
Nuclease-free water	Up to 50 μL

The reaction was stored at -20°C or immediately used for downstream experiments .

Results:

The efficiency of phosphorylation of DNA ends was evaluated by downstream experiments.