Week 2: Validation of Recombineering-edited

Introduction

After obtaining strepR colonies from the last lab exercise, the first step to validate the expected AA→CC two-nucleotide conversions in the edited cells is to isolate genomic DNA from strepR colonies, followed by PCR amplification of the target site, agarose gel electrophoresis of the PCR product, and purification of the remaining DNA, for sequencing and analysis. Bacterial genomic DNA isolation often involves centrifugation to collect the cells, then suspending the pellet in a buffer, followed by adding a lysis solution to break the cell wall and membrane.²⁹ RNase A and proteinase K are added to destroy RNA and degrade bacterial proteins. The lysis step usually proceeds for approximately 1 hour. The DNA, not RNA or proteins in the lysate, is then bound to a silica-based binding column, from which the genomic DNA is eluted. The entire isolation process takes several hours to complete.

Fortunately, E. coli is Gram-negative with a thin cell wall—this enables individual colonies to be used directly for PCR amplification without the need for genomic DNA isolation because, during so-called colony-PCR, bacteria are lysed to release their DNA during the initial heating step before the amplification.³⁰ Colony-PCR combines bacterial genomic DNA isolation and PCR into one step. Due to its simplicity and the requirement of a minute quantity of bacterial cells, colony-PCR has been used to identify bacterial transformants (bacterial colonies containing recombinant plasmid constructs) in DNA cloning, taxonomy screening, and diagnostics while preserving most of the colony for subsequent analysis.

For this week’s lab, colony-PCR amplification of the target site in the rpsL of strepR colonies will be conducted. The PCR product is then electrophoresed in an agarose gel to validate the presence of the expected 370 bp PCR product (Figure 3-4). The remaining PCR product is purified and outsourced for DNA sequencing, followed by sequence analysis. Results should confirm the two-nucleotide substitution (AA→CC) in the recombineering-edited cells, which prevents the S12 protein from binding to streptomycin and allows the cells to survive in the antibiotic selection medium.

Procedure

A. Colony PCR

Use filter pipette tips for all the following steps.

1. Obtain three 0.2 ml PCR tubes and label them with your group number and the corresponding control and treatments. Each group should set up three PCRs using colonies they obtained. For example, group 1, control (1C-1, 1C-2, 1C-3) if colonies are available, sense ssODN (3S-1, 3S-2, 3S-3), and antisense ssODN (5A-1, 5A-2, 5A-3).
2. Set up 50 µl PCR reactions as shown below.
   

   *We use Phusion™ High-Fidelity DNA Polymerase (2 U/µl)
   PCR reaction	(50 µl)
   Sterile distilled water	26 µl
   5X buffer with MgCl2	10 µl
   10 mM dNTPs	1 µl
   Forward primer (rpsL-F) (2.5 µM) (5′-atggcaacagttaaccagct-3′)	6 µl (0.3 µM final)
   Reverse primer (rpsL-R) (2.5 µM) (5′-ccttaggacgcttcacgc-3′)	6 µl (0.3 µM final)
   Taq Polymerase with proofreading*	0.5 µl
   Total	50 µl

3. Touch a single colony each from control (C) (if there is any), ssODN (S), and antisense ssODN (A) plates without digging into the agar. Do not try to scoop up the colony.
4. Submerge the tip in the 50 µl PCR reactions and rub it against the wall of the PCR tube.
5. Amplify the samples in a thermal cycler.
   • 1 cycle of 98°C for 30 sec
   • 35 cycles of 98°C for 10 sec, 72° C for 30 sec, and 60°C for 15 sec
   • Final extension of 72°C for 5 min

B. Agarose Gel Electrophoresis

While waiting for the PCR, you will prepare an agarose gel using the steps shown below.

1. Seal a gel tray with masking tape and place a comb on it.
2. Make a 1.8% agarose gel by adding 0.81 g of agarose in 45 ml of 1X TAE (adding 0.9 ml of 50X TAE and 44 ml of RO water) in a 125 ml flask and heat it in a microwave oven until the agarose completely melts.
3. Add 4.5 µl GelRed and mix well by swirling the flask gently (avoid creating bubbles in the flask).
4. Pour the agarose solution into the gel tray and allow it to set.
5. Place the gel tray in the electrophoresis box, add 300 ml of 1X TAE buffer, and remove the comb.
6. After the completion of colony-PCR, add 2 µl of 6X DNA loading buffer and 5 µl of each of the finished PCR products to a strip of parafilm. Note: We used 2 instead of 1 µl of DNA loading buffer, because your pipettes can only measure ≥ 2 µl.
7. Mix by pipetting up and down on the parafilm and then load the entire aliquot of each of your three samples into each well.
8. Load 5 µl of 100 bp DNA ladder.
9. Electrophorese the samples at 100V constant voltage until the bromophenol blue is approximately 3/4 of the distance through the gel (~1 hr.)
10. Visualize the DNA fragments under the BioRad GelDoc Go Imaging System or similar device, photograph, and estimate the size of your PCR product on the gel.

C. Purification of PCR Products with a PCR Clean-up Kit

All centrifugation steps are conducted at 17,900x g (13,000 rpm) in a microcentrifuge at room temperature. 

1. Add a 1:1 volume of Binding Buffer to the remaining completed PCR mixture (e.g., for 100 µl of the reaction mixture, add 100 µl of Binding Buffer).
2. Mix thoroughly by pipetting. The color of the mix should become a yellowish hue. The yellow color of the solution indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 µl of 3 M sodium acetate, pH 5.2 solution, and mix. Optional: If the DNA fragment is ≤ 500 bp (our case), add a 1:2 volume of 100% isopropanol (e.g., 100 µl of isopropanol should be added to 100 µl of PCR mixture combined with 100 µl of Binding Buffer). Mix thoroughly.
3. Transfer up to 800 µl of the solution from step 1 (or optional step 2) to the GeneJET purification column. Centrifuge for 30–60 sec. Discard the flow-through.
4. Add 700 µl of Wash Buffer (diluted with the ethanol as described by the manufacturer) to the column.
5. Centrifuge for 30–60 sec, discard the flow-through and place the purification column back into the collection tube.
6. Centrifuge the empty GeneJET purification column for 1 min to remove any residual wash buffer. Note: This step is essential as residual ethanol in the DNA sample may inhibit subsequent reactions.
7. Transfer the GeneJET purification column to a clean 1.5 ml microcentrifuge tube.
8. Add 35 µl of Elution Buffer to the center of the GeneJET purification column membrane and centrifuge for 1 min.
9. Transfer each of the eluted samples into each of the 1.5 ml tubes in ice, measure OD for the quantity and purity of the purified PCR products, and record the results in Table 3-4.
10. Prepare 20 µl of your PCR product (20 ng/µl) and 1 µl per reaction of 5 µM rpsL-R primer (5′-ccttaggacgcttcacgc-3′) for DNA sequencing. Place your samples in the rack designated by your instructor for outsourcing DNA sequencing. Note: The ratio of 260/280 of your sample’s OD reading must be between 1.8 and 2.0. Otherwise, the sequencing reaction will likely fail or produce poor results due to contamination or improper concentration.

Data Analysis and Discussion

1. Describe how to make a 45 ml of 2% TAE agarose gel using 50X TAE buffer.
2. a.   Without making the standard curve, please use the marker lane to estimate the approximate size of DNA bands in the gel. Are the size(s) expected?
   2. Would you expect to see any difference in the sizes of PCR products between the control and recombineering edited bacterial colonies? Explain.
3. a.   Create a multiple DNA sequence alignment to check DNA sequences at the target site of the entire class’s PCR products obtained. (For instruction, recall steps for the previous Data Analysis and Discussion in Module 2 Week 5).
   2. Based on the results from your answer in (a), did the experiment work? Explain.
4. Read next week’s lab and watch the following videos: “CRISPR gene editing will transform cancer” by the Verge, “But what is CRISPR-cas9?” by Powerhouse, and “What is the future of gene editing?” by Al Jazeera, prior to answering question 5.