Experiments

Goal

Preparation of a stock of Luria-Bertani broth.

Protocol

Luria-Bertani broth was prepared using tryptone (Cat. No. 4864701, SERVA), yeast extract (Cat. No. 2454002, SERVA), and sodium chloride (Cat. No. 3978102, SERVA). The pH was adjusted to 7.0 using sodium hydroxide (Cat. No. 6771, Carl ROTH). The final composition of the broth was as follows:

Component Quantity:

• Yeast extract: 5 g/L
• Tryptone: 10 g/L
• Sodium chloride: 10 g/L
• Ultrapure water: to x 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.

Goal

Preparation of LB agar plates with antibiotic.

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.

Goal

The gBlocks were resuspended in IDTE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) to a final concentration of 100 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 100 ng/μL.

Results

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

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 (Cat. No. C3019, New England Biolabs) 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 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. 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 selection agar plate.

Goal

Colony picking of individual DH10β E. coli (Cat. No. C3019, New England Biolabs) bacterial colonies transformed with our construct 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.

Goal

Minipreps 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 (ND-1000 Spectrophotometer), 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.

Goal:

A maxiprep was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Protocol

DNA plasmids were prepared using the QIAGEN ® Plasmid 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 5804 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 (ND-1000 Spectrophotometer), 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.

Goal

Cell-free transcription-translation (TXTL) reaction was performed to in vitro-express tyrosinase for enzyme activity testing under the control of a T7 promoter.

Protocol

TXTL was performed using proprietary E.coli cell extract and reaction buffer B from Invitris GmbH. The composition of each sample was as follows:

Buffer B: 7 μL
Cell extract: 5 μL
T7 Polymerase: 0.16 μL
DNA: 2.7 μL (to the concentration of 20 nM)

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

Results

The efficiency of TXTL was evaluated by downstream experiments.

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 (Bio-Rad ChemiDoc MP).

Results

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

Goal

A gel extraction was performed to isolate and purify a specific DNA fragment from agarose gel for downstream applications.

Protocol:

Following gel electrophoresis, DNA bands of interest were excised from the gel under UV transillumination (BIORAD) using a clean scalpel. The excised gel slices were transferred to a 1.5 mL microcentrifuge tube and weighed to determine the amount of gel present. DNA fragments were then isolated and purified from gel using the Monarch® DNA Gel Extraction Kit Protocol (NEW ENGLAND Biolabs, NEB #T1020) following modified manufacturer’s instructions. For this, the gel slices were first dissolved in 4 volumes of Monarch Gel Dissolving Buffer (NEW ENGLAND Biolabs) at 50 °C. The mixture was then transferred to a provided column placed in a collection tube. The column was centrifuged at 13500 × g (Eppendorf 5804 R) for 1 min to allow the DNA to bind to the column. The flow-through was discarded, and the column was washed twice with 200 μL of DNA Wash Buffer (NEW ENGLAND Biolabs) at 13500 × g (Eppendorf 5804 R) for 1 min each to remove impurities and contaminants. Purified DNA was eluted into a 1.5 mL microcentrifuge tube by adding 10 μL of elution buffer to the column matrix, incubating at room temperature for 1 min, and centrifuging the column for 1 min at 13500 × g (Eppendorf 5804 R). DNA concentration was determined using a (ND-1000 Spectrophotometer), and the DNA samples were stored at -20 °C.

Results

The extraction procedure yielded DNA.

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 (Eppendorf 5804 R). 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 (Eppendorf 5804 R). The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g (Eppendorf 5804 R) 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 (Eppendorf 5804 R). DNA concentration was determined using a NanoDrop (ND-1000 Spectrophotometer), 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.

Goal

To confirm the nucleotide sequences of our constructs, 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.

Goal

To confirm the nucleotide sequences of a plasmid, whole plasmid sequencing was performed by using Genewiz, Azenta services.

Protocol

DNA was sequenced using a Whole Plasmid sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 25 μL of purified plasmid at a concentration of 50 ng/μL The tube was 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.

Goal

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments 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 (ND-1000 Spectrophotometer). 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
Nuclease-free water: Fill up to 25 μL

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

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.

Goal

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments 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 (ND-1000 Spectrophotometer). The final composition of the reaction mixture is defined as follows:

Components:
2X Platinum™ SuperFi™ II PCR Master Mix: 10 μL
Forward + Reverse Primers (pre-mixed at 5 μM each): 0.5 μM
Template DNA: 10 ng
Nuclease-free water: Fill up to 20 μL

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

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.

Goal

Restriction digest of the construct with enzyme(s), for downstream cloning of other constructs.

Protocol:

Restriction digest was carried out using the enzyme(s) 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.

Goal

Ligation of our constructs to assemble futher constructs.

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
T4 DNA Ligase Buffer (10x) 2 μL
Vector DNA 50 ng (0.020 pmol)
Insert DNA 37.5 ng (0.060) 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.

Goal

Gibson assembly was performed to assemble the vector and inserts to produce a final construct.

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 around 150 ng
• Nuclease-free Water to 20 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.

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 manufacturers 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.

Goal

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

Protocol

Dephosphorylation of 5′ ends of DNA was carried out using Quick CIP (M0525S, New England Biolabs) according to the manufacturers protocol. Template DNA was supplied in amount of 1 pmol of DNA ends (about 1 μg of a 3 kb plasmid) and combined with 2 μL of rCutSmart (B7204, New England Biolabs) and 1 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 2 minutes.

Component Volume:

• DNA: 1 pmol of DNA ends
• rCutSmart buffer: 2 μL
• Quick CIP: 1 μL
• Nuclease-free Water: to 20 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.

Goal

Purification of proteins carrying a Strep-Tag for downstream experiments.

Protocol

Purification of proteins was carried out using a Strep-Tactin®XT 4Flow® high capacity Spin Column Kit (iba Lifesciences) according to the manufacturers protocol at 4 °C. Resuspend Strep-Tactin®XT 4Flow® high capacity resin and pipet 100 μL of the 50% suspension into a spin column. Centrifuge the sample (maximum speed, 5 min, 4 °C) to remove aggregates. Apply 500 μL sample to the spin column, close the column lid and incubate at room temperature with constant movement for 30 min. 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. Collect the washing fraction for SDS-PAGE analysis and place the spin column into a new reaction tube. 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 time. At least 80% of the target protein will be in the first elution fraction.

Results

We successfully purified proteins that carried a Strep-Tag.

Goal

Purification of proteins carrying a Strep-Tag for downstream experiments.

Protocol

Purification of proteins was carried out using a Strep-Tactin®XT 4Flow® high capacity column (iba Lifesciences) according to the manufacturers protocol at 4 °C.
Samples with a volume of 200 - 450 µL were applied to the column. If needed, samples were diluted in Wash Buffer (iba LifeSciences) to achieve at least 200 µL sample volume. The sample was allowed to pass through the column via gravity flow, the flowthrough was collected in a separate eppendorf tube. The column was washed twice with 1 mL Wash Buffer each. Wash 1 and Wash 2 were collected in separate eppendorf tubes. Next, 1 mL Elution Buffer was applied to the column, then the column was sealed with the lid and incubated for 10 minutes. Elution 1 was collected in a separate eppendorf tube. Afterwards, the column was washed three times with 0.5 mL Elution Buffer, with every single elution step being collected in a separate eppendorf tube. The column was regenerated using Buffer R, made out of NaOH, with two regeneration steps of 1 mL each. Lastly, the column was washed with Wash Buffer until the column turned colorless again. To store the column, it was coated with 1 mL Wash Buffer and closed with the lid.

Results

The efficiency of purification was evaluated by downstream experiments.

Goal

The BCA was performed to determine the total protein concentration in the sample.

Protocol

Preparation of BSA standard

Pipette 25 µL of each standard or sample replicate into microplate well. Add 200 µL of WR to each well & mix plate throughly on a plate shaker for 30 seconds with Multichannel pipette. Cover plate & incubate at 37 °C for 30 minutes in incubator. Equilibrate plate to room temperature. Measure absorbance at 562 nm on plate reader. 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. 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.

Results

Protein concentrations of the samples were determined from the standard curve, with absorbance values proportional to protein content.

Goal

Monitoring of tyrosinase activity with high sensitivity.

Protocol

Monitoring of tyrosinase activity was carried out using a Tyrosinase Assay Kit (Merck, MAK550) according to the manufacturers protocol. Tyrosinase standard solutions were made by diluting a stock solution (2000 U/mL) 1:5 using Assay Buffer to achieve a final concentration of 400 U/mL (T1), followed by 1:2 serial dilutions to produce the remaining serially diluted tyrosinase standards (T2-T7). The working solution was prepared by adding 20 µL tyrosinase substrate stock solution (50X) and 20 µL tyrosinase enhancer into 960 µL Assay Buffer to achieve a 1:50 dilution. For the assay reaction, 50 µL of each standard, blank (Assay buffer), and test samples were added in triplicates into separate wells of a 96-well plate. Then, 50 µL of tyrosinase substrate working solution was added to each well containing a standard, blank, or test sample to make the total assay volume of 100 µL per well. The well plate was incubated at room temperature for 30 - 60 minutes. Absorbance was measured with an absorbance plate reader at OD of 510 nm.

Results

Tyrosinase activity was detected in the test samples, with absorbance values increasing proportionally to enzyme concentration. Sample activity was quantified based on the standard curve, allowing accurate determination of enzymatic activity in each test sample.

Goal

Analyze protein content in the sample.

Protocol

Protein samples were separated by SDS–PAGE using 12% resolving and 6% stacking gels prepared in Tris–HCl/SDS buffer. Electrophoresis was carried out at 200V in Tris–glycine running buffer until clear separation of bands was achieved.

Results

Distinct protein bands corresponding to the expected molecular weights were observed after Coomassie staining. The band pattern reflected the protein composition of the sample, with clear separation indicating proper electrophoretic resolution.

Goal

Confirm the presence of a specific protein in the sample.

Protocol

Protein samples were separated by SDS–PAGE using 12% resolving and 6% stacking gels prepared in Tris–HCl/SDS buffer. Electrophoresis was carried out at constant voltage in Tris–glycine running buffer until clear separation of bands was achieved.

Following electrophoresis, proteins were transferred onto nitrocellulose membranes using a wet transfer system in Towbin buffer (25 mM Tris, 192 mM glycine, 20% methanol, 0.0375% SDS) at 400 mA for 1.5 h. Membranes were briefly rinsed with distilled water and blocked in 5% (w/v) non-fat milk dissolved in TBS–T (20 mM Tris–HCl, 150 mM NaCl, 0.1% Tween-20, pH 7.6) for 1 h at room temperature with gentle agitation.

Blocked membranes were incubated with primary antibodies diluted in TBS–T for 1 h at room temperature or overnight at 4 °C, followed by three washes in TBS–T (5 min each). HRP-conjugated secondary antibodies diluted in TBS–T were applied for 1 h at room temperature, and membranes were again washed three times for 5 min each.

Protein bands were visualized using enhanced chemiluminescence (ECL) detection reagents and imaged with the ImageLab system.

Results

Distinct protein bands corresponding to the expected molecular weights were/were not observed after chemiluminescent detection. The target protein was detected in the relevant sample lanes, confirming successful expression and transfer. The molecular weight marker exhibited well-resolved bands, indicating proper electrophoretic separation and transfer efficiency.

Goal

To separate proteins in their native, non-denatured form to assess their oligomeric state.

Protocol

Protein samples were analyzed using native polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions. 12% gels were prepared in Tris–HCl buffer without SDS. Protein samples were mixed with native loading dye lacking SDS, β-mercaptoethanol and glycerol.

Electrophoresis was carried out for 20 mins at 50V and 2 hours at 200V in Tris–glycine running buffer (25 mM Tris, 192 mM glycine, pH 8.3) at 4 °C. The run continued until the dye front reached the bottom of the gel, ensuring clear band separation.

Following electrophoresis, gels were stained with Coomassie Brilliant Blue to visualize proteins. Staining was performed for 1 h with gentle shaking, followed by destaining in water until clear background and distinct protein bands were obtained. Gels could alternatively be used for activity assays or imaging in UV for detection of fluorescently labelled proteins.

Results

Distinct bands corresponding to native protein species were observed after staining. Migration patterns reflected differences in protein size, charge, and conformation, allowing assessment of oligomerization and protein integrity under native conditions.

Goal

Splitting HEK293T cell line from a T25 flask to the next passage number.

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 T25 flask was inoculated with the 1 mL cell suspension to 9 mL pre-warmed DMEM. Cells were grown at 37°C with 5% CO₂ and 90% relative humidity.

Results

HEK293T cell line from a T25 flask was split.

Goal

Calculating cell suspension concentration and assessing the quality of the suspension.

Protocol

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet. Cells were counted and analyzed for viability using Countess™ II automated cell counter with two replicates, and the average of both measurements was taken.

Results

Countess™ II automated cell counter results:

Goal

Seeding Nunc™ MicroWell™ 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate (Thermo Scientific™) for further confocal analysis of HEK293T cells.

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:

Formulas for seeding calculation

Goal

Introduction of our plasmids and controls 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% CO₂ and 90% relative humidity.

Results

The efficiency of transfection was evaluated by downstream experiments.

Goal

Confocal imaging of HEK293T cell culture. Cells were transfected with our plasmids and controls to analyze our experiment.

Protocol

Culturing media was decanted from the 96-well microscopic slides from ibidi. Cells were washed using 300 µL of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) to remove any unviable cells from the slide. Fresh 300 µL of Dulbecco’s modified Eagle medium F12 (DMEM) containing [Hoechst/Second stain] was supplied to the cell culture for identification of the cell nucleus. Afterwards, cells were incubated for 10 minutes at 37 °C with 5% CO₂ and 90% relative humidity. The 96-well slide was then imaged using CellInsight™ CX7 High Content Analysis Platform from Thermo Scientific™.

Results

The aquired pictures were evaluated based on the transfection plan.