Lab Notebook

Our complete laboratory notebook documenting daily activities, observations, and experimental progress throughout the EPIC System development.

Lab Timeline Overview

This notebook chronicles our journey from initial plasmid isolation through the complete characterization of our dCas9-Dam system and fluorescence assays. Each month's work is organized below with detailed daily entries.

Monthly Lab Activities

📅 June 2025

9th June, Monday

Primary Isolation and Purification of pdCas9 plasmid

Culturing of cells with pdCas9

• We streaked the stab culture of cells having the pdCas9 plasmid (obtained from Addgene) on agar plates
• We incubated the streaked plates at 37°C for 16 hours

10th June, Tuesday

Primary Isolation and Purification of pdCas9 plasmid

Culturing of cells with pdCas9

• We supplemented the liquid culture with Chloramphenicol and incubated it at 37°C at 180 rpm for 16 hours

11th June, Wednesday

Primary Isolation and Purification of pdCas9 plasmid

Isolation and purification of pdCas9 from the liquid culture:

• We performed Miniprep using MN kit (low-copy plasmid protocol )
• We checked the concentration and purity of the purified pdCas9 plasmid on Nanodrop (~220 ng/ul)

🔍

• We Performed gel electrophoresis to confirm the plasmid.
• Ran the gel(1% agarose) for 45 min at 100V
• We observed a diffused band near the 10kb band on the ladder. (Our plasmid is 9.3kb)

2) Cloning of gRNA target sequence into the dCas9 Plasmid

• Annealing of oligonucleotides

13th June, Friday

2) Cloning of gRNA target sequence into the dCas9 Plasmid

Digestion of pdCas9 plasmid with BsaI

• We used 20 Units BsaI enzyme and 1ug plasmid in 20uL of reaction volume.
• The reaction was incubated for 16 hours at 37°C

15th June, Sunday

2) Cloning of gRNA target sequence into the dCas9 Plasmid

Purification of the linearized plasmid from the reaction mixture (Removal of salts, enzymes and small DNA fragments)

• We used the MN PCR clean-up kit for spin column purification

🔍

(NucleoSpin Column; silica membrane column for binding to DNA and collection tube)

• We checked the concentration and purity of the linearized plasmid in Nanodrop : 5 ng/uL

The concentration of the DNA obtained was very low and not satisfactory to proceed further.

1) Repeat Isolation and Purification of pdCas9 Plasmid

Transformation of competent cells with pdCas9

• We transformed XL1 Blue cells by Heat shock Method

Culturing of Transformed cells

• We spread transformed cells on agar plates with Chloramphenicol and Tetracycline antibiotics

16th June 2025, Monday

1) Isolation and Purification of pdCas9 Plasmid

Culturing of Transformed cells

• We inoculated colonies from the plate in LB broth
• We incubated the liquid culture for 16 hrs at 37°C in shaking incubator

2) Cloning of gRNA target sequence into the dCas9 Plasmid

Repeated digestion of pdCas9 with BsaI

• We repeated the digestion with a larger reaction volume to get a better concentration of the linearized plasmid after purification.

17th June 2025, Tuesday

2) Cloning of gRNA target sequence into the dCas9 Plasmid

Purification of the linearized plasmid from the reaction mixture (Removal of salts, enzymes and small DNA fragments).

• We used the same kit again to purify the linearized plasmid with modifications to improve DNA elution:
• Incubate the column at 70°C after adding Elution buffer for 5 minutes
• Elute twice
• We checked the concentration and purity of the linearized plasmid on Nanodrop (50ng/ul). It was satisfactory to proceed further

Ligation of linearized plasmid and annealed oligos.

• We set up the ligation reaction for inserting the oligo with BsaI overhangs in the linearized pdCas9 plasmid.
• The molar ratio of plasmid to oligos in the ligation mix was nearly 1:600
• We incubated the ligation mix at 4°C for one day.

1) Isolation and Purification of pdCas9 Plasmid

Isolation and purification of pdCas9 from the liquid culture:

• We repeated miniprep for 5 aliquots of 6mL liquid using the MN Kit
• We checked the concentration and purity of the purified pdCas9 plasmid in Nanodrop.
• We got satisfactory concentration and purity for 4 out 5 aliquots
• We stored the plasmid at -20°C

🔍

18th June 2025, Wednesday

2) Cloning of gRNA target sequence into the dCas9 Plasmid

Transformation and culture of cells with recombinant dCas9 Plasmids

• Transformed the colonies using heat shock with the ligation mix
• We plated on Chloramphenicol+Tetracycline agar plate and incubated overnight at 37°C

19th June 2025, Thursday

2) Cloning of gRNA target sequence into the dCas9 Plasmid

Colony PCR for screening recombinant plasmids using repurposed primers

We expect a 230 bp amplicon if our gRNA insert is successful.

One primer was specific to the plasmid. The other primer was one of the single-stranded oligos of the insert (These primers were originally meant for a different purpose. They were not ideal for PCR)

• We prepared cell suspensions of 12 colonies and streaked a Chloramphenicol agar plate with the cell suspensions along with a negative control(a colony with the original dCas9) and a non-template control
• We prepared PCR reactions using the cell suspensions as the templates.

Thermal cycling conditions for PCR: 30 cycles:

• 94°C for 1 min
• 52°C for 1 min
• 72°C for 30sec
• 72°C for 5 min

3) Isolation and Purification of the dnaAP2-GFP plasmid

Suspension of lyophilized Plasmid

The pET-blank plasmid(AmpR) with our custom dnaAP2-gfp sequence was shipped in the lyophilized form (~1600 ng).

• We suspended the DNA in 8 μL of Tris-HCL Buffer

Transformation with the GFP plasmid.

• We transformed XL1 Blue competent cells using heat shock method

Culturing of transformed cells

• We plated transformed cells on Ampicillin+Tetracycline agar plate and incubated the plate at 37°C for 16 hours.

20th June, 2025 Friday

2) Cloning of gRNA target sequence into the dCas9 Plasmid

Agarose Gel Electrophoresis for Colony PCR

• We ran the PCR reaction mix on 1% agarose gel
• We observed broad and intense bands of around 100-200 bp bands in all the samples as well as the negative controls.

We made the mistake of loading too much DNA in each well, hence the bands did not resolve properly. 1st gel image- ladder followed by 7 samples 2nd gel image- ladder followed by samples-8,9,10,11,12 and then non template controls

🔍

🔍

🔍

Polyacrylamide Gel Electrophoresis for Colony PCR

We ran some of the samples (the ones that have the possibility of having our amplicon) on native 8% polyacrylamide gel to get better resolution

All the samples and the negative control had bands of size less than 100bp, probably primers. We did not get any positive colonies The below gel shows the ladder followed by the samples-8,11,12 and then non template control

🔍

3) Isolation and Purification of the dnaAP2-GFP plasmid

Culturing of Transformed Cells

• We prepared three 6 mL liquid cultures ( with Ampicillin + Tetracycline) of cells transformed with GFP plasmid.
• We incubated the liquid cultures at 37°C overnight.

21st June, Saturday

3) Isolation and Purification of the dnaAP2-GFP plasmid

Isolation and purification of the plasmid from liquid culture

• We performed mini-prep using MN Kit
• Checked the concentration of the isolated plasmid in Nanodrop

The concentration for 2 of the 3 plasmid samples was satisfactory considering the fact that the cultures were not fully grown (there was an issue with the incubator)

🔍

2) Cloning of gRNA target sequence into the dCas9 Plasmid

Gel Electrophoresis for Linearized Plasmid

• We ran the digested dCas9 plasmid along with the intact dCas9 plasmid on 0.6% agarose gel at 100V.
• We adjusted the volume of each sample for loading such that each well has nearly 100 ng of DNA

For the digested dCas9, we observed a diffused band (undigested plasmid) and a thin and precise band, close to 9.3kb (linearized plasmid). The fraction of linearized plasmid was very low.

🔍

The lack of digestion was potentially because the enzyme was expired

22nd June, Sunday

3) Isolation and Purification of the dnaAP2-GFP plasmid

Gel electrophoresis for plasmid confirmation

We ran the three GFP plasmid samples (6.2kb) isolated yesterday on the gel shown above. The three rightmost lanes showed a diffused band lying between 5-7kb confirming the plasmid.

🔍

23rd June, Monday

2) Cloning of gRNA target sequence into the dCas9 Plasmid (Attempt2)

Digestion of pdCas9 plasmid with BsaI

We repeated digestion with a higher enzyme concentration, less amount of DNA and longer incubation period. We were trying to get a higher yield of digested plasmid.

24th June, Tuesday

2) Cloning of gRNA target sequence into the dCas9 Plasmid (Attempt2)

Purification of the linearized plasmid from the reaction mixture

We used the MN PCR clean-up kit to purify the linearized plasmid with the modified DNA elution protocol We checked the concentration and purity of the linearized plasmid using the nanodrop. We obtained a satisfactory concentration of 32ng/ul

Ligation of linearized plasmid and annealed oligos

We tried two different plasmid to insert ratios in the ligation mix (1:600 and 1:60) and prepared a negative ligation control without the insert (i.e. annealed oligos) We incubated the three reaction mixes at 4°C overnight

25th June, Wednesday

2) Cloning of gRNA target sequence into the dCas9 Plasmid (Attempt2)

Transformation of cells with ligation mix and culture on agar plates

We transformed three competent cell aliquots with the 1:600 , 1:60 and the control ligation mix by the heat shock method We plated transformed cells on Chloramphenicol +Tetracycline agar plates. We incubated the plates at 37°C overnight.

The plates next day:

Control

🔍

1:60

🔍

1:600

🔍

We observed no significant difference in the number of colonies between control and both of the ligated plates implying lack of colonies with recombinant plasmid due to low digestion. We decided not to proceed with colony pcr since the probability of obtaining a recombinant colony from the plate is very small.

🧬 July 2025

2nd July, Wednesday

4) Cloning of dCas9-Linker-Dam plasmid

Culturing of E.coli cells

• We prepared 6 mL liquid culture of XL1 Blue cells and incubated the liquid cultures overnight at 37°C

3rd July, Thursday

4) Cloning of dCas9-Linker-Dam plasmid

Isolation of genomic DNA from E.coli culture

• We used the Qiagen DNeasy Blood and Tissue Kit for isolation of genomic DNA
• We checked the concentration of the isolated genomic DNA on Nanodrop ( got ~180 ng/uL )

🔍

4th July, Friday

4) Cloning of dCas9-Linker-Dam plasmid

PCR Amplification of Dam gene from E.coli genome

Suspension of Lyophilized Oligos

• Oligo 3 and oligo 4 were shipped by IDT in lyophilized format so we prepared 100uM stocks of oligo 3 and oligo 4 by suspending them in TE buffer

We diluted the primers and genomic DNA (template)to get the desired working concentrations

We used Takara's high fidelity Primestar GXL Polymerase for PCR

We designed the reaction mix and PCR thermal conditions according to the Primestar GXL User Manual(standard 3-step PCR product protocol for 10kb product)

PrimeSTAR® GXL DNA Polymerase

PCR Thermal Settings:

Initial denaturation step : 94°C for 2 min (genomic DNA) Amplification (30cycles): Denaturation: 98°C for 10 sec
Annealing: 60°C for 15 sec
Extension: 68°C for 1 min (1min/kb) Hold at 4°C

5th July, Saturday

4) Cloning of dCas9-Linker-Dam plasmid

PCR Amplification and Linearization of pdCas9 Plasmid

Suspension of Lyophilized Oligos

• Oligo 1 and oligo 2 were shipped by IDT in lyophilized format so we suspended them in TE buffer to prepare 100uM stock solutions

We diluted the primers and plasmid (template) to get the desired concentrations

We used Takara's high fidelity Primestar GXL Polymerase.

We designed the reaction mix and PCR thermal conditions according to the Primestar GXL User Manual and Takara's webpage on PCR Optimization.

PrimeSTAR® GXL DNA Polymerase user manual Optimizing your PCR Takara webpage

We are using the "Rapid 3-Step PCR protocol for 10kb product" from the GXL user manual. Our amplicon is ~9.3 kb. Rapid protocol uses twice the amount of polymerase for faster extension.

PCR Thermal Conditions:

Initial denaturation step : 94°C for 2 min

Amplification (30cycles):

• Denaturation: 98°C for 10 sec
• Annealing: 55°C for 15 sec
• Extension: 68°C for 3 min (10sec /kb)

Final Extension: 68°C for 2 min Hold at 4°C

Agarose Gel Electrophoresis for confirmation of PCR Products

• We ran the PCR products (Dam and linearized dCas9) on 0.7% agarose gel to confirm the presence of our desired amplicons
• Wells 4 and 5 were loaded with 3ul and 1 ul of the Dam PCR product (~900bp) respectively and wells 7 and 8 were loaded with 3ul and 1ul of dCas9 PCR product (~9.3kb) respectively.

The bands on the gel (~900bp for Dam amplicon and ~9.3kb for the linearized plasmid) confirmed the presence of our desired amplicons and absence of non-specific PCR amplification.

🔍

🔍

7th July, Monday

4) Cloning of dCas9-Linker-Dam plasmid

Purification of PCR Products from PCR reaction mix

• We used the MN PCR clean-up kit for purification of the PCR products and checked the concentration of purified PCR products on nanodrop: 27 ng/ul for Dam and 88 ng/ul for pdCas9

In-Fusion Cloning Reaction

• We prepared an in-fusion cloning reaction mix containing the in-fusion mix, linearized plasmid and the insert (Dam) in 1:2 molar ratio.
• We incubated it at 50°C for 15 minutes

Transformation of Competent cells with in-fusion reaction mix

• We transformed XL Blue competent cells by heat shock method.
• We spread the transformed cells on Tetracycline+Chloramphenicol agar plate and incubated the plate at 37°C overnight.

Next day, we had about 7-8 colonies on the plate

9th July, Wednesday

4) Cloning of dCas9-Linker-Dam Plasmid

Colony PCR for Screening of Recombinant Colonies

We repurposed oligo 3 and oligo 4 as primers for colony PCR. They are insert-specific chimeric primers(i.e. their 5' ends are complementary to Dam but 3' ends are complementary to the plasmid)

There is a possibility of getting false positive results due to amplification of the endogenous Dam gene.

• We made suspensions of 4 colonies from dCas9-Dam plate.
• We also picked 2 colonies from the dCas9 plate to serve as negative control (NC1 and NC2).
• We boiled all cell suspensions at 98°C for 10 min to lyse the cells.
• The cell suspensions were used in PCR along with non-template control (NC3)
• We performed touchdown PCR to increase specificity of primers to the Dam insert in plasmid over endogenous Dam

PCR Thermal Settings (Touchdown/stepdown PCR):

Initial denaturation: 94°C for 2 min

8 cycles:

• Denaturation: 94°C for 30sec
• Annealing: 70°C for 30 sec (reduce by 2°C every 2 cycles)
• Extension: 72°C for 1 min

22 cycles:

• Denaturation: 94°C for 30 sec
• Annealing: 62°C for 30 sec
• Extension: 72°C for 1 min

Final extension: 72°C for 3 min Hold at 4°C

Gel Electrophoresis of colony PCR products

• We ran the PCR products on 0.7% agarose gel for 40 minutes

The gel shows the ladder followed by 4 samples and then non template controls

🔍

We observed intense bands(~900 bp) in all lanes including negative controls NC1 and NC2 due to amplification of endogenous Dam, as we feared Hence, colony PCR was inconclusive.

Culturing of Transformed Cells

• We made liquid culture (Chloramphenicol+Tetracycline) for 4 colonies from the dCas9-Dam plate We incubated the liquid cultures overnight at 37°C

10th July, Thursday

4) Cloning of dCas9-Linker-Dam Plasmid

Isolation of recombinant (hopefully) plasmids

We performed miniprep to isolate the plasmids We checked the concentration of isolated plasmids on Nanodrop

🔍

Concentrations and purity of all 4 four plasmid isolates were satisfactory.

16th July, Wednesday

2) Cloning of gRNA target sequence into the dCas9 Plasmid +4) Cloning of dCas9-Linker-Dam Plasmid

Test Digestion of pdCas9 plasmid with BsaI +Screening of dCas9-Dam recombinant plasmids

We acquired fresh NEB BsaI- hfV2 enzyme that had been used last year by the iGEM team. To check enzyme activity, we decided to perform a test digestion of pdCas9 with BsaI.

We digested the four dCas9-Dam plasmids( S1 to S4) as well as one original dCas9 plasmid (NC). We wanted to use the test digestion as a screening test for dCas9-DAM recombination as well.

We prepared five 10ul digestion mixes. Four with dCas9-Dam plasmids and one with pdCas9
We incubated the digestion mixes at 37°C for 30 min

We loaded the reaction mixes on 1% agarose gel (~100ng DNA in each well) along with the ladder, undigested dCas9 (UNC) and dCas9-Dam (U1) plasmids. We ran it at 100V for 1 hour.

The gel shows ladder followed by 3 samples and then U1 and UNC

🔍

We observed nearly 100% digestion which means the BsaI enzyme that we acquired had retained most of its activity.

We could not distinguish between the 9.3kb (pdCas9 and 10.2 kb(pdCas9-Dam) bands due to poor resolution. Hence the screening for recombination was not successful

18th July, Friday

4) Cloning of dCas9-Linker-Dam Plasmid

Screening of recombinant plasmids using restriction digestion

Original dCas9 plasmids will give 8kb and 1.35kb bands whereas the recombinant dCas9-Dam plasmids are supposed to give 8kb, 1.09kb and 1.15 kb bands after complete digestion with BamHI

We digested the four dCas9-Dam plasmids (S1 -S4) along with two dCas9 plasmids as controls.

We ran the samples on 1% agarose gel

🔍

We observed very faint bands around 10kb in size. This is the singly undigested product. Most of the plasmid was undigested.

Again, the screening results were inconclusive due to incomplete digestion.

20th July, Sunday

4) Cloning of dCas9-Linker-Dam Plasmid

PCR Screening of recombinant plasmids

We decided to perform PCR again, but this time we decided to use isolated plasmids as template to avoid amplification of Dam gene present in genomic DNA which had given us false positive results last time

We diluted the plasmid samples to get the desired working concentration

For negative control (NC), we used the original pdCas9 plasmid (1 ng), and for positive control (PC), we used E.coli genome (8ng)

PCR Thermal Settings:

• Initial denaturation: 94°C for 2 min

• 28 cycles:

  • Denaturation: 94°C for 30 sec

  • Annealing: 64°C for 30 sec

  • Extension: 72°C for 1 min

3. Final extension: 72°C for 3 min
4. Hold at 4°C

Gel Electrophoresis of PCR products

We ran the PCR products on 1% agarose gel for about 45 min

The gel shows the positive control, non template control, followed by 4 samples and then the ladder

🔍

We observed a band of ~900 bp in 3 out of 4 plasmid samples indicating Dam amplification. This band was absent in the negative control. Hence, screening was finally successful with S1,S2 and S4 positive for dCas9-Dam recombinant plasmids

Plasmids and primers sent for sequencing

We prepared the positive dCas9-Dam samples (S1, S2 and S4) for sequencing: 20ul at 100 ng/ul

We prepared 40ul of ~10uM of each of the primers we had used for Dam PCR (Oligo 3 and Oligo 4)

Each of the plasmids will be sequenced once using primer P1 and once using primer P2

29th July 2025, Tuesday

4) Cloning of dCas9-Linker-Dam Plasmid

Sequencing Results:

Two of the four plasmids that we had sent for sequencing were positive for recombination (S2 and S4) But we could not verify the complete reading frame of dCas9-Linker-Dam because of poor resolution at the junctions between linker-dam and Dam-plasmid ( because the primers P1 and P2 bind at the junctions).

30th July 2025, Wednesday

2) Cloning of gRNA target sequence into the dCas9 Plasmid

Annealing of oligonucleotides

Digestion of pdCas9 and dCas9-Dam plasmids with BsaI

We digested the original dCas9 plasmid and the recombinant dCas9-Dam plasmids (S2 and S4)

We incubated the three digestion mixes at 37°C for 1 hour followed by heat inactivation at 80°C for 20 min.

Purification of the linearized plasmids from the digestion mixtures

We used the MN kit to purify the linearized plasmid from the three reaction mix

The purity of all three samples was poor (a sharp unwanted peak around 220-230nm was observed) but we still decided to proceed with ligation.

🔍

S2: 92 ng/ul S4: 60 ng/ul dCas9: 85 ng/ul

Ligation of linearized plasmid and annealed oligos

We took 1:20 plasmid to insert (annealed oligos) ratio in the ligation mix We prepared the ligation mix for all three digestion mixes. We also prepared a control ligation mix (without annealed oligos) We incubated the ligation mixes overnight

🔬 August 2025

1st August, Friday

4) Cloning of dCas9-Linker-Dam Plasmid

Transformation with Ligation Mix and Culturing of Transformed Cells

• We used the heat shock protocol for transformation of 4 aliquots XL1 Blue cells with the four ligation mixes
• We plated the transformed cells on agar plates (Chloramphenicol+Tetracycline)
• We incubated the plates overnight at 37°C

The next day, we observed ~150 colonies on the control plate as well as on the S2 (dCas9-Dam) and dCas9 plates. The S4 (dCas9-Dam) plate had only about 10 colonies.

Since the control plate had nearly as many colonies as the recombinant ones, we decided to repeat the cloning again.

4th August 2025, Monday

6) Repeat Isolation of dCas9-Dam Plasmids

Transformation and Culturing of Transformed cells

• We transformed XL1 Blue competent cells with dCas9-Dam plasmid (S2 and S4) by heat shock method
• We plated the transformed cells on two agar plates (Chloramphenicol+Tetracycline)
• We incubated the plates overnight at 37°C

7) Testing of Co-transformation (dnaAP2-gfp and dCas9 plasmids):

Co-Transformation and Transformation of Control groups:

• We co-transformed one aliquot of XL1 Blue competent cells with 200ng each of GFP and dCas9-Dam(S2) plasmids by heat shock method
• For controls, we transformed 2 aliquots with GFP and 1 aliquot with dCas9-Dam (S2) - 200ng

Culturing of Transformed Cells:

Single Transformation controls:

• GFP transformed cells on Amp+Tet agar plate
• S2 transformed cells on Chlor+Tet agar plate

We plated the (GFP+dCas9-Dam) co-transfected cells on (Amp+Chlor) agar plate.

Negative controls:

• GFP cells on Amp+Chlor plate
• S2 cells on Amp+Tet plate

We incubated all the plates for 16 hours at 37°C.

The next day, we observed small translucent colonies of the XL1 Blue strain (the ones we used) and big opaque colonies which were due to contamination (probably fungal colonies since it was growing in all three antibiotics: Amp, Chlor, Tet)

All the plates were contaminated, the contamination could have happened during transformation or the competent cell aliquots that we prepared could be contaminated.

XL1 Blue colonies (Translucent colonies):

• Gfp(6kb) cells on Amp+Tet: ~ 10* 10^3
• S2(10kb) cells on Chlor+Tet: ~ 3-4* 10^3
• Gfp+S2 cells on Amp+Chlor: ~ 0.5-0.6* 10^3
• Gfp cells on Amp+Chlor: nil
• S2 cells on Amp+Tet: nil

If we only look at the XL1 Blue colonies, we got the expected results:

• Top row (Single Transformation): Had lots of colonies. Gfp transformation was more efficient as compared to dCas9-Dam
• Middle row (Co-transformation): Had 6-fold less and 20-fold less colonies as compared to single transformation with gfp and dCas9-Dam plasmids respectively.
• Bottom row (Negative controls): Had no colonies(except satellite colonies which were growing around the fungal colonies)

The results are very much in line with expected trends for heat-shock transformations.

• Large plasmid = lower transformation efficiency
• Co-transformation = probability drop due to dual entry requirement

🔍

6th August 2025, Wednesday

6) Isolation of dCas9-Dam Plasmids

Culturing of Transformed Cells:

• We inoculated the four colonies from the transformed plate in 6ml LB broth each supplemented with Tet and Chlor antibiotics
• We incubated the liquid cultures for 16hrs at 37°C, 180rpm

7) Testing of Co-transformation (dnaAP2-gfp and dCas9 plasmids):

Culturing of Co-transformed Cells:

• We carefully picked out two colonies from the co-transformed plate while avoiding the colonies of the contamination species.
• We inoculated the two colonies in 6ml LB broth each supplemented with Amp+Chlor antibiotics
• We incubated the liquid cultures for 16hrs at 37°C, 180rpm

7th August 2025, Thursday

6) Isolation of dCas9-Dam Plasmids:

Plasmid extraction from Liquid culture

• We performed miniprep for dCas9-Dam plasmid extraction
• We measured the concentration of the the plasmids on Nanodrop

In order: S2_a, S2_b, S4_a, S4_b

🔍

The concentrations were lower than desired and the A260/A230 were also off.

7) Testing of Co-transformation (dnaAP2-gfp and dCas9 plasmids):

Plasmid extraction from Liquid culture

• We performed miniprep using the MN kit to extract plasmids from the co-transformed culture.
• We measured the concentration of the the plasmids on Nanodrop

319 ng/ul: S2+gfp_a

228 ng/ul: S2+gfp_b

The concentrations of both the samples were satisfactory. The A260/A230 ratios were a little off.

8th August 2025, Thursday

6) Isolation of dCas9-Dam Plasmids:

BsaI digestion and gel electrophoresis for plasmid confirmation

• Rnx Mix for Digestion:

  • 10X CutSmart Buffer : 1ul

  • BsaI-hfV2 : 0.2ul (4 units)

  • Plasmid : ~1 to 2 ul (200 ng)

  • NF H20: make to 10ul

• We digested the four dCas9-Dam samples (S2_a, S2_b, S4_a, S4_b) and dCas9 (control).

• We prepared the master mix first, aliquoted 8ul of MM into pcr tubes and added the plasmids at last.

• We incubated the digestion mixes at 37°C for 30 min

• We ran the five digestion mixes on 1% agarose at 100V for 1 hour. ( 50ng DNA = 2.5ul dig mix in each well)

• We observed ~10kb bands in all four dCas9-Dam samples and a ~9kb band in the dCas9 control. This confirmed the presence of dCas9-Dam plasmid in all our samples.

7) Testing of Co-transformation (dnaAP2-gfp and dCas9 plasmids):

BsaI digestion and gel electrophoresis for plasmid confirmation

The dCas9-Dam plasmid is expected to give a 10.2kb band after digestion with BsaI. The dnaAP2-gfp plasmid is expected to give a 6.2kb band after single digestion and 1.5kb and 4.6 kb bands after double digestion.

• We digested the plasmid samples from co-transformation with BsaI
• We ran the two digestion mixes on 1% agarose at 100V for 1 hour.
• We observed four bands (~10kb, ~6kb, ~4.5kb, 1.5kb) in both the dual plasmid samples, thus confirming the presence of both dCas9-Dam and dnaAP2-gfp plasmids.( These are referred to as GFP below)

Co-transfarmation was successful! The gel shows ladder followed by S2( 2samples), S4( 2 samples), dCas9 and then S2+GFP(2 samples)

🔍

9th August 2025, Thursday

2) Cloning of gRNA target sequence into the dCas9/ dCas9-Dam Plasmid

Golden gate assembly

• We realised that gRNA cloning into pdCas9 is suitable for golden gate assembly with BsaI restriction enzyme and T4 ligase.
• We used a 10:1 insert to vector ratio.
• We prepared three ligation mix (S2, S4 dCas9-Dam plasmids and pdCas9 plasmid) and a control ligation mix without the annealed oligos
• We incubated the reaction mix for 45min followed by 10 min at 60°C.

Transformation with golden gate mix

• We transformed four 200ul aliquots of XL1 Blue cells with golden gate reaction mix by the heat shock method.

Culturing of Transformed cells

• The transformed cells were plated on four agar plates (S2, S4, pdCas9 and Control) supplemented with Chloramphenicol+Tetracycline antibiotics.
• We incubated the plates at 37°C for 16 hours.

The next day, we observed colonies on all four plates. The control plate (dCas9) and the recombinant plate (dCas9) nearly had the same number of colonies which was a cause of concern but we decided to proceed with screening.

8) dnaAP2-GFP Fluorescence Assay for Dam(-) cells.

Transformation of Dam(-) competent cells with gfp plasmids and culturing of transformed cells

• We used the heat shock protocol given by NEB to transform 50ul Dam(-) cells.
• We plated the transformed Dam(-) strain on Amp+Chlor agar plate and incubated overnight at 37°C ( the cell strain was already resistant to Chlor)

The next day, we observed only about 5 colonies on the transformation plate and no colonies on the control (untransformed) plate. The low transformation efficiency was concerning. It could be due to the Dam(-) genotype. We also realised that the Dam(-) competent cell strain that we procured from NEB is Chlor resistant so we cannot use Chlor-resistance as a selection marker for transforming with our dCas9/dCas9-Dam plasmid.

18th August 2025, Monday

2) Cloning of gRNA target sequence into the dCas9 Plasmid

Colony PCR for screening of recombinant colonies

We used primers P1 and G for PCR screening. P1 is specific to the plasmid and G is specific to the insert. (This time the primers were designed specifically for PCR.) The recombinant colonies are expected to give a band of size 220 bp.

• We picked 3 colonies from each ligation plate (dCas9, dCas9-Dam_S2, dCas9-Dam_S4) and suspended them in 10 ul NF H2O.
• We also picked 1 colony from the control plate we had prepared earlier, and suspended it in 10 ul NF H2O to serve as negative control
• We boiled 2ul of each cell suspension at 98°C for 10 min to lyse the cells for pcr
• We stored rest of the cell suspension at 4°C for preparing liquid of positive colonies
• We used the lysed cell suspensions as templates for pcr reaction

For non-template negative control, we used the remaining master mix as pcr reaction mix:

Thermal cycler settings:

Initial Denaturation: 94°C for 2min 30 Cycles:

• Denaturation: 94°C for 30 sec
• Annealing: 55°C for 30 sec
• Extension: 72°C for 1 min
• Hold: 4°C

Next day, we realised that we had put the PCR tubes in the wrong wells in the PCR machine which means that the temperature cycling could have been sub-optimal.

9) dnaAP2-GFP Fluorescence Assay for wild-type Dam(+) cells.

Culturing of GFP transformed cells and untransformed cells

• We prepared 2 aliquots of liquid culture of XL1 blue cells containing the GFP plasmid supplemented with Ampicillin+Chlor antibiotics
• We also prepared one liquid culture with untransformed XL1 blue cells.
• We incubated the three liquid cultures overnight at 37°C, 180rpm

19th August 2025, Tuesday

9) GFP Fluorescence Assay for native Dam(+) cells.

Preparation of samples for microplate assay

• We pelleted 1mL of each liquid culture.
• We washed the cell pellet with PBS buffer twice and finally resuspended it in PBS buffer.

The cell pellet with gfp (left) was visibly greener as compared to the non-gfp cell pellet (right). This indicates the expression of the dnaAP2 promoter.

🔍

20th August 2025, Wednesday

2) Cloning of gRNA target sequence into the dCas9 Plasmid

Gel Electrophoresis of Colony PCR product for Screening

• We ran the PCR reactions on 1.5% agarose gel at 100V for 45 min

• We observed a lot of non-specific amplification in dC2, S2_1 and S4_2 indicating that they were negative for recombination

• dC1, S2_2 and S2_3 and MM had no bands at all. Hence, these samples were inconclusive for recombination

• dC3(marked with the green circle) and S4_1 had a sharp band of the right size(220bp) without any non-specific bands. The band in dC3 was intense while the one in S4_3 was faint. But nonetheless, S4_3 and dC_3 seemed to be positive for our recombinant plasmid.

• The first gel shows the ladder followed by dC1, dC2,dC3, S2_1, S2_2 and then the negative control

• The second gel shows the ladder followed by S2_3, S4_1, S4_2, S4_3 and then the negative control

🔍

🔍

9) GFP Fluorescence Assay for native Dam(+) cells

Fluorescence and Cell Density Measurements using Microplate Reader

• We prepared 1:1 and 1:4 dilutions of cell suspensions
• We put duplicates of each dilution (and undiluted) of each cell suspension (one non-gfp and two gfp) in the microplate.
• We measured OD600 and fluorescence intensities (gain 76)

Normalized GFP readings of the three samples:

🔍

More diluted samples showed higher gfp/OD600 ratios which we later found out was due to a phenomenon called fluorescence quenching. Hence it is not possible to compare samples at different dilutions even after normalization.

We decided to stick to 1:4 (cell culture to buffer) dilution henceforth to minimize the effect of fluorescence quenching.

22nd August 2025, Friday

2) Cloning of gRNA target sequence into the dCas9 Plasmid

Repeated Colony PCR and Gel Electrophoresis for screening of Recombinant plasmids

• We got a clear positive result for the gRNA insert in dCas9 plasmid but only a doubtful positive result for one dCas9-Dam with gRNA insert sample
• So we repeated the same protocol as on 18th August for colony PCR for colonies from dCas9-Dam plates (S2 and S4).
• We ran the PCR products on agarose gel
• We did not get any positive colonies this time.

Since we are short of time, we decided to send the plasmids from the positive colonies we got in the previous screening for sequencing

26th August 2025, Tuesday

2) Cloning of gRNA target sequence into the dCas9 Plasmid

Liquid Culture of Cells with Recombinant Plasmids

• We used the stored cell suspensions to prepare liquid culture for colonies that tested positive and the ones that were indeterminate in the pcr screening.
• We prepared 12mL liquid culture for positive samples dC_3 and S4_3
• We prepared 6mL liquid culture for indeterminate samples S2_2 and S2_3
• We supplemented all the liquid cultures with Chlor+Tet antibiotics and incubated overnight at 37°C, 180rpm

8) dnaAP2-GFP Fluorescence Assay for Dam(-) cells.

Transformation of Dam(-) competent cells with gfp plasmids and culturing of transformed cells.

• We repeated the transformation of Dam(-) E.coli with dnaAP2-gfp plasmid using the same heat shock protocol we used on 9th August.
• We plated the transformed cells on Amp+Chlor agar plate.
• We also plated untransformed Dam(-) cells on a Chlor agar plate (i.e. without selection for plasmid)
• We incubated the plates overnight at 37°C

The next day, we observed only 2-3 colonies on the Amp+Chlor plate and thousands of colonies and matting on the Chlor plate. This indicates low transformation efficiency which could be due to loss of competency during transportation

27th August 2025, Wednesday

2) Cloning of gRNA target sequence into the dCas9 Plasmid

Plasmid isolation from liquid cultures

• We performed miniprep using MN kit for liquid cultures DC_3, S4_3, S2_2, S2_3
• We checked the plasmid concentrations on Nanodrop:

🔍

28th August 2025, Thursday

2) Cloning of gRNA target sequence into the dCas9 Plasmid

Preparation of plasmids and primers for sequencing

• We prepared the plasmids that we had isolated yesterday and primers to send for sequencing.

Primer P1: 15 uM 40uL Primer P2: 15 uM 20uL Primer O4: 15 uM 20 uL

• Primer P1 will be used for sequencing the target sequence region and the plasmid backbone-Dam junction.
• Primer P2 will be used for sequencing the dCas9-Dam junction.
• Primer O4 will provide high resolution for the Dam sequence that is outside the range of P1 and P2.
• The plasmids and primers were packed and sent for sequencing.

🧪 September 2025

1st September 2025, Monday

8+9) dnaAP2-GFP Fluorescence Assay for Dam(-) cells and Dam(+) cells

Culturing of Dam(-) cells and XL1 blue cells Dam(+) with gfp

• We prepared 6mL liquid culture each from two colonies of Dam(-) cells that were transformed with the dnaAP2-gfp plasmid.
• We supplemented the liquid cultures with Amp+Chlor antibiotics. (The plate appeared to be contaminated since the colonies were unusually large)
• We prepared 6mL liquid culture from one colony of untransformed Dam(-) cells and supplemented it with Chlor antibiotic.
• We prepared 6mL liquid culture each from two colonies of Dam(+) XL1 Blue cells that were transformed with the dnaAP2-gfp plasmid.
• We supplemented it with Amp+Tet antibiotics.
• We incubated the liquid cultures for about 15hrs 40 min at 37°C, 180 rpm

2nd September 2025, Tuesday

8+9) dnaAP2-GFP Fluorescence Assay for Dam(-) cells and Dam(+) cells

Preparation of samples for microplate assay

• We pelleted 1 mL each of the five liquid cultures.
• We washed the cell pellets twice with PBS buffer and resuspended them in PBS buffer

The XL1 blue cell pellet with gfp was strongly green and the Dam(-) cell pellet with gfp was very faintly green as compared to the untransformed cell pellet (right).

Fluorescence and Cell Density Measurements using Microplate Reader

• We prepared 1:4 dilutions ( 1 part cell suspension + 4 parts buffer) of each sample.
• We put triplicates of each sample in the microplate.
• We measured 0D600 absorbance and fluorescence intensity (gain 75)
• Averaging, Blanking and normalization were performed on the readings

Final GFP expression readings:

🔍

🔍

Gfp expression in Dam(-) cells was reduced nearly five-fold as compared to Dam(+) cells. (Matches with our visual observations)

5th September 2025, Friday

2) Cloning of gRNA target sequence into the dCas9 Plasmid

Sequencing Results

• dC_3 (dCas9): insertion successful

• S4_3 (dCas9-Dam): negative

• S2_3 (dCas9-Dam): negative

• S2_2 (dCas9-Dam): sequencing issue

5) Cloning of dCas9-Linker-Dam Plasmid

Sequencing Results:

The sequence at plasmid-Dam and Dam-linker-dCas9 junctions was confirmed to be completely correct for the S4 sample. Henceforth we will be using S4 as the dCas9-Dam plasmid

8th September 2025, Monday

2) Cloning of gRNA target sequence into the dCas9-Dam Plasmid

Transformation with golden gate mix

• Since gRNA cloning in dCas9 plasmid was successful, there is a good chance that it was successful for dCas9-Dam as well because we performed cloning on both simultaneously.

• To screen more colonies, we decided to repeat the transformation with the golden gate ligation mix we had prepared on 9th August for S4 (dCas9-Dam).

• We transformed XL1 Blue cells with about 4ul of golden gate mix using the heat shock method

• We plated the transformed cells on Chlor+Tet agar plate and incubated overnight at 37°C

• Next day we observed about 20-30 colonies on the plate

9th September 2025, Tuesday

2) Cloning of gRNA target sequence into the dCas9-Dam Plasmid

Colony PCR for screening of recombinant plasmids

• Used the same protocol that we had used on 18th August for screening colonies from the plate we made yesterday

Gel Electrophoresis for colony PCR products

• Same protocol as followed on 18-19 August.
• Due to errors in gel preparation and running the gel, electrophoresis was unsuccessful.

10) Competent Cell Stock of Dam(-) Cells with dnaAP2-gfp Plasmid

Transformation of Dam(-) cells with gfp Plasmid

• NEB Dam(-) cells were transformed with the gfp plasmid using the heat shock method mentioned in the 9th August entry.
• Transformed cells were plated on Amp+Chlor agar plates and incubated overnight at 37°C.

Next day, we only had one colony on the plate (poor transformation efficiency as we had seen earlier also)

10th September 2025, Wednesday

2) Cloning of gRNA target sequence into the dCas9-Dam Plasmid

Repeat Colony PCR for screening of recombinant plasmids

• Used the same protocol that we had used on 18th August for screening colonies from the plate we made on 8th September.
• We screened 12 colonies and used the dC_3 liquid culture for the positive control since it was confirmed positive by sequencing.

10) Competent Cell Stock of Dam(-) Cells with dnaAP2-gfp Plasmid

Repeat Transformation of Dam(-) cells with gfp Plasmid

• NEB Dam(-) cells were transformed with the gfp plasmid using the heat shock method mentioned in the 9th August entry.
• Transformed cells were plated on Amp+Chlor agar plates and incubated overnight at 37°C.

The next day, we had two colonies on the plate.

11th September 2025, Thursday

2) Cloning of gRNA target sequence into the dCas9-Dam Plasmid

Gel Electrophoresis for Colony PCR products

• Same protocol as followed on 19th August
• The below gel shows the ladder followed by 6 samples and then the positive template

🔍

None of the colonies we tested turned out to be positive for guide RNA cloning.

Culturing of cells having dCas9 Plasmid with gRNA insert

• We prepared three 6mL liquid culture aliquots of XL1 Blue cells with the dC_3 plasmid (dCas9 plasmid that was confirmed to be positive for guide RNA target insert).
• The liquid cultures were supplemented with Chlor+Tet antibiotics and incubated for about 18 hours at 37°C, 180rpm.

Competent Cell Stock of Dam(-) Cells with dnaAP2-gfp Plasmid

Culturing of Dam(-) cells transformed with gfp Plasmid

• Prepared three 6mL liquid culture from the three colonies of Dam(-) gfp cells supplemented with Amp+Chlor antibiotics
• Incubated overnight at 37°C, 180rpm

The next day ~1.5ml of each liquid culture was stored at 4°C

12th September 2025, Friday

2) Cloning of gRNA target sequence into the dCas9-Dam Plasmid

dC_3 Plasmid isolation

• Performed spin-column mini-prep for the liquid culture prepared yesterday using the MN kit with the protocols mentioned earlier
• We got very plasmid low concentration after isolation.

13th September 2025, Saturday

10) Competent Cell Stock of Dam(-) Cells with dnaAP2-gfp Plasmid

Competent cells of dam negative cells with dna2-gfp was made and stored at -80°C. Spread 100 ul of this to check for contamination

2) Cloning of gRNA target sequence into the dCas9-Dam Plasmid

Culturing of cells having dCas9 Plasmid with gRNA insert

• We prepared liquid culture aliquots of XL1 Blue cells with the dC_3 plasmid for repeating plasmid isolation

• The liquid cultures were supplemented with Chlor+Tet antibiotics and incubated for about 18 hours at 37°C, 180rpm.

14th September 2025, Sunday

2) Cloning of gRNA target sequence into the dCas9-Dam Plasmid

Repeat dC_3 Plasmid isolation

Performed spin-column mini-prep for the liquid culture prepared yesterday using the MN kit with the protocols mentioned earlier

This time we obtained a better plasmid concentration after isolation

15th September

2) Cloning of gRNA target sequence into the dCas9-Dam Plasmid

Annealed the grna (4 and 10) following the protocol used earlier Since there was no T4 ligase in the lab, we could not proceed further with cloning

16th September

2) Cloning of gRNA target sequence into the dCas9-Dam Plasmid

• Obtained T4 ligase from ug lab and performed cloning( cloned the grna-4 and grna-10 in the dcas9-dam plasmid)

• Made slight modifications to the protocol used before-After digestion and ligation of the grna with the plasmid

  • Split the volume in two and performed pcr clean up for one half and did not perform pcr clean up for the other half

  • Used close to 8 ul of the pcr product for the transformation

• then we did the transformation of the XL1 blue cells with the above plasmid and the old ligation mix

18th September

2) Cloning of gRNA target sequence into the dCas9-Dam Plasmid

Set a colony pcr for the colonies but didnt get any positive results

🔍

20th September

2) Cloning of gRNA target sequence into the dCas9-Dam Plasmid

Repeated colony pcr but did not get an positive results( even the positive control- dcas9+grna didnt have any bands implying a problem with the pcr itself)

🔍

22nd September

2) Cloning of gRNA target sequence into the dCas9-Dam Plasmid

After discussion with other phd scholars and the professor, we realised that the taq polymerase was not functioning properly so changed the polymerase and set another pcr reaction. This time we obtained a band in the positive control at the expected length but did not get a band in any of the colonies indicating the gRNA cloning was not successful

26th September

2) Cloning of gRNA target sequence into the dCas9-Dam Plasmid

• Used the following modified protocol for grna cloning:

  1. Plasmid digestion-

  • Reaction mixture:

    • plasmid 600 ng/ul
    • bsa1-1 ul
    • cut smart- 2 ul
    • total- 20 ul

  • Incubate at 37°C for 2 hours

• Performed a PCR clean-up

• Measured the concentration of the digested plasmid and obtained very low values of around 5ng/ul which was not sufficient for ligation so decided to redo the digestion

🎯 October 2025

3rd October

2) Cloning of gRNA target sequence into the dCas9-Dam Plasmid

• Set up a digestion reaction again- performed the same protocol as 26th September but decided to modify the initial starting concentration of the plasmid( started with 1000 ng/ul) to improve plasmid concentration after digestion
• Obtained concentrations of 130 g/ul, 20 ng/ul and 15 ng/ul after pcr cleanup which was sufficient to proceed with ligation
• Since the A260/280 ratio was around 1.5 instead of 1.8 we decided to do another restriction enzyme digestion just incase
• Ligated the colonies with the gRNA using the following protocol:

Reaction mixture:

• Plasmid: insert must be in 1:6 ratio so using the NEB ligation calculator we got the amounts to be approximately 129ug of insert per ug of plasmid
• T4 DNA ligase: 1ul
• T4 DNA ligase buffer: 2ul
• Nuclease free water: To make the total volume to 20 ul
• Did an overnight incubation at 16°C and then stored the ligation mixture at 4°C

4th October

2) Cloning of gRNA target sequence into the dCas9-Dam Plasmid

• Used the heat shock protocol to transform the XL1 colonies with the ligation mixture and plated the cells on lb agar with chloramphenicol and tetracycline

5th October

2) Cloning of gRNA target sequence into the dCas9-Dam Plasmid

• Tested the colonies using PCR and obtained colonies with expected band size of 200 bp which could indicate positive cloning so we sent those colonies for sequencing