The following fragments all use the same PCR amplification system, with the difference being that the templates are different.

A. Cas9-λ-Red fragment: Use the plasmid pEcCas9-λ-Red as a template.

B. Cas9-op-λ-Red fragment: Use the plasmid pEcCas9-op-λ-Red as a template.

C. p15A fragment: Use the synthetic plasmid containing p15A as a template.

D. ColE1 fragment: Use the synthetic plasmid containing ColE1 as a template.

E. gntT homology arm: Use the plasmid pEcgRNA-gntT as a template.

F. lacZ homology arm: Use the plasmid pEcgRNA-lacZ as a template.

The reaction system for PCR amplification is shown in the table below:

 

The reaction procedure for PCR amplification are shown in the table below:

Notice: Repeat step 2 to step 4 for 28 times.

 

1. Preparation of agarose gel

1) Add 100 mL 1× TAE buffer and 1 g agarose to an flask, and place it in a microwave oven on high for 2 min.

2) Take it out after the solution turns transparent and shake it evenly.

3) Add 2 μL nucleic acid dye GelRed to the molten agarose.

4) Assemble the gel tray and insert comb into mold.

5) Pour the liquid agarose into the mold, avoiding air bubbles.

6) Wait for solidification of the gel at room temperature.

7) Carefully remove comb and transfer solidified gel to electrophoresis tank.

8) Completely submerge gel in 1´ TAE buffer.

2. Loading samples

1) Add DNA loading buffer into samples to dye them blue, then load into wells sequentially.

2) Add 10 μL of Trans15K DNA Marker into a lane.

3. Electrophoresis

1) Place wells at the negative electrode, and cover tank.

2) Turn on the power, and run the agarose gel at 180 V for 20 min.

4. Visualizing DNA bands

1) Turn off power, and remove gel.

2) Place the gel under the Ultra Slim LED Illuminator to visualize the results.

3) Take out the gel, and put it under gel-imaging system to save data.

 

1. Excise the DNA band of interest under UV light using a clean scalpel.

2. Transfer the gel slices into centrifuge tubes, and measure the weight of the gel slices.

3. Add 3 times of sample volume of Buffer B2 into the centrifuge tubes, and mix until gel completely dissolved.

4. Transfer the solution into the absorption column, and centrifuge at 9000 ×g for 30 sec. Pour off the filtrate in the collection tube.

5. Add 500 μL of Wash Solution and centrifuge at 9000 ×g for 30 sec. Pour off the filtrate in the collection tube.

6. Repeat wash step.

7. Centrifuge the empty column at 9000 ×g for 1 min.

8. Place the absorption column into a clean 1.5 mL centrifuge tube, add 15-40 μL Elution Buffer to the center of the absorption membrane, and centrifuge at 9000 ×g for 1 min. Store the DNA solution in the tube.

 

For the recombinant plasmids p15A-Cas9-λ-Red, p15A-Cas9-op-λ-Red, and ColE1-Cas9-op-λ-Red, since the upstream and downstream primers used previously for fragment amplification contained homology arms, the following fragments can be homologously recombined using the ClonExpress II One Step Cloning Kit:

 

The homologous recombination reaction system is as follows:

* X/Y is calculated according to the formula according to the manual.

Incubate at 37 °C for 30 min; cool to 4 °C or place on ice immediately.

 

1. Plasmid pEcgRNA was digested with BsaI to generate linearized pEcgRNA carrying the overhangs 5′-TAGT-3′ and 5′-AAAC-3′.

The digestion system is shown in the table below:

The enzyme digestion system is incubated at 37 ℃ for 3 h.

 

2. Single-strand oligonucleotides comprising 4 nt of the overhangs and 20 nt of target-specific sequences (gntT or lacZ) were annealed to form dsDNA.

The annealing system is shown in the table below:

 

3. The reaction mix was incubated at 95 °C for 5 min, and the temperature was then gradually reduced by 5–10 °C/min. The reaction was finally held at 16 °C for 10 min.

4. The annealed dsDNA was then diluted 200-fold and 1 µL of the diluted DNA was ligated to 1 µL of BsaI-linearized pEcgRNA in a mixture of T4 ligase buffer and T4 ligase for 1 h at 16 °C.

 

1. Add 5 μL of plasmids into 50 μL of E. coli DH5α competent cells, respectively. Incubate the mixture on ice for 30 min.

2. Heat shock at 42 °C for 90 sec, and put the mixture to the ice for 4-5 min.

3. Add the LB culture into the tubes to 1 mL, and shake the tubes at 37 ℃, 220 rpm for 30-60 min.

4. Centrifuge it at 9000 ×g for 1 min, remove 800 μL of supernatant, and resuspend the cells in the remaining LB culture.

5. Spread 50 μL cells onto LB agar plates containing spectinomycin or chloromycetin and incubate overnight at 37 ℃.

 

1. Place 50 μL of Lysis Buffer for Microorganism to Direct PCR in a sterilized microtube. Pick a single colony with a sterilized toothpick or lance tip, place it in the microtube, and stir a few times before removing it.

2. After heat denaturation at 80 °C for 15 min, centrifuge at low speed and take 1~5 μL of supernatant after lysis as PCR template.

The PCR reaction system is shown in the table below:

Reagent	Volume (μL)
Lysis supernatant	1
Premix Taq (Ex Taq Version 2.0)	10
Forward Primer (10 μM)	1
Reverse Primer (10 μM)	1
sterilized water	7

 

The PCR reaction condition is shown in the table below:

Step	Temperature (˚C)	Time (sec)
1	94	600
2	94	30
3	55	30
4	72	120
5	72	300
6	4	¥

Notice: Repeat step 2 to step 4 for 28 times.

 

3. Take positive transformants and add 3 mL LB culture medium containing spectinomycin or chloromycetin, shake bacteria, and incubate at 37 ℃ at 220 rpm for 12-16 h.

4. Inoculate the positive transformants in 3 mL LB culture medium containing spectinomycin or chloromycetin at a 37 ℃ shaker with 220 rpm for 12-16 h.

5. Extraction the plasmids from overnight cultures.

 

1. Equilibrate the adsorption column

1) Add 500 μL of Buffer S to the adsorption column and place a collection tube below it.

2) Centrifuge at 12,000 ×g for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

2. Transfer the E. coli culture into a 2 mL centrifuge tube, centrifuge at 12,000 ×g for 2 min to collect the bacteria. Discard the supernatant and retain the precipitate.

3. Add 250 μL of Buffer SP1 to the 2 mL centrifuge tube, and thoroughly suspend the precipitate.

4. Add 250 μL of Buffer SP2, and gently invert the tube 5-10 times to mix the solution.

5. Add 350 μL of Buffer SP3 and mix the solution by gently inverting the tube 5-10 times.

6. Centrifuge at 12,000 ×g for 5-10 min. Transfer 700 μL of the supernatant to the adsorption column, centrifuge at 8,000 ×g for 30 sec, and discard the filtrate in the collection tube.

7. Add 500 μL of Wash Solution, centrifuge at 9,000 ×g for 30 sec, and discard the filtrate in the collection tube.

8. Repeat wash step.

9. Centrifuge the empty column at 9,000 ×g for 1 min.

10. Place the column into a clean 1.5 mL centrifuge tube, and add 25 μL of Elution Buffer to the center of the adsorption membrane. Incubate for 1 min at room temperature, centrifuge at 9,000 ×g for 1 min, and keep the DNA solution in the tube.

 

1. Cultivate the E. coli Nissle 1917 strain overnight in LB medium at 37 °C, 200 rpm/min.

2. Inoculate 50 mL LB medium on the second day at 1% inoculation volume.

3. When the OD₆₀₀ of the freshly inoculated culture reaches 0.6-0.8, ice-bath the culture for 20 min to fully cool.

4. Centrifuge the culture in 50 mL centrifuge tubes at 4000 rpm/min and 4 °C for 10 min. Discard the supernatant and quickly return the tubes to ice.

5. Resuspend the cell pellet in ice-cold sterile ddH₂O or 10% glycerol by pipetting up and down on ice. Centrifuge again at 4000 rpm/min and 4 °C for 10 min. Discard the supernatant and quickly return the tubes to ice.

6. Repeat the wash step 1-2 times.

7. According to the number of transformation samples, add ice-cold sterile deionized water to the tubes at 100 μL per sample, and resuspend the cell pellet.

8. Transfer 100 μL of resuspended cells per sample into electroporation cuvettes on ice.

9. Add approximately 100 ng of Cas9 plasmids (p15A-Cas9-λ-Red, p15A-Cas9-op-λ-Red, and ColE1-Cas9-op-λ-Red) to be transformed, mix well, and ensure the cells-plasmid mixture is between the two metal electrodes inside the cuvette.

10. Wipe the exterior metal electrodes of the cuvette dry and electroporate at 1.8 kV, 200 Ω, 25 μF. Immediately add 900 μL LB without antibiotics and resuspend by pipetting. Transfer the cuvette contents into a 1.5 mL tube.

11. Recover the electroporated cells by shaking at 37 °C for 1 h. Plate on LB agar with chloramphenicol and incubate overnight at 37 °C.

12. Pick transformants and culture overnight at 37 °C, 200 rpm/min in liquid LB with chloramphenicol.

13. Inoculate chloramphenicol-containing LB medium on the second day at 1% inoculation volume. Add L-arabinose to a final concentration of 10 mM.

14. Repeat steps 1-12 to electrotransform pEcgRNA (pEcgRNA-gntT and pEcgRNA-lacZ) and homology arms into competent cells containing the Cas9 plasmid.

 

1. Transfer E. coli Nissle 1917 culture to 50 mL centrifuge tubes and centrifuge at 8000 rpm for 5 min. Discard supernatant and pool all cell pellets.

2. Add 15 mL bacterial protein extraction buffer (100 mM NaCl, 10 mM EDTA, pH 8.0) to each tube. Resuspend pellets by pipetting up and down.

3. Place tubes in a rack, submerge probe tip of ultrasonic cell disruptor into tube. Position tube and close door. Turn on power and sonicate each tube with 5 cycles of 2 sec on, 2 sec off at 80 W power.

4. Centrifuge at 8000 rpm for 5 min after 4 treatments.

5. Carefully collect supernatant as crude protein extract into new tubes and store at 4 °C.

 

1. Column assembly

Fix the column on iron stand. Unscrew top adaptor and seal bottom outlet with a clamp. Add deionized water to remove air bubbles. Prepare 2-5 mL slurry of 20% ethanol-equilibrated Chelating Sepharose Fast Flow resin and pack into column with a glass rod against inner wall. Open outlet to let resin settle. Resin volume 2-5 mL. Reattach top adaptor and seal inlet/outlet with tubing.

2. System setup

(1) Peristaltic pump: Turn on the power, press start, and set flow rate to 15 rpm (2 mL/min) with arrow keys. Fill the tubing with deionized water.

(2) Connect pump outlet tubing to column inlet. Connect column outlet to UV monitor inlet. Connect UV monitor outlet to the fraction collector.

(3) UV monitor: Turn on power, adjust ABS sensitivity knob and zeroing knob.

(4) Fraction collector: Turn on power, reset, set tube position via manual control, then set collection parameters (e.g. 120 sec per tube). Press auto to begin timed collection.

(5) Record UV absorbance data on computer to generate chromatograms.

3. Column equilibration

(1) Wash ethanol-preserved column with 5× bed volumes of deionized water to remove ethanol.

(2) Pass 2× bed volumes of 0.2 M NiSO₄ through column. Observe phenomena.

(3) Wash with 10× bed volumes of deionized water. Equilibrate with 5× bed volumes of 0.1 M PBS with 5 mM imidazole.

4. Sample loading

First, take 1 mL from 20 mL lysate supernatant as total protein control before chromatography. Load clarified lysate supernatant onto column at 2 mL/min flow rate. Collect 1 mL fractions, with highest UV peak as flowthrough. Wash with equilibration buffer until UV absorbance plateau is reached. Load 15 mL sample.

5. Washing

Wash with 20× bed volumes of PBS with 15 mM imidazole (pH 8.0) to remove weakly bound contaminants. Collect flowthrough and analyze.

6. Elution

Elute bound proteins with 10 mL PBS containing 500 mM imidazole. Collect 1 mL fractions and identify the tube with the highest UV absorbance peak.

 

1. Prepare BSA standards of known concentrations.

2. Add standards and protein samples to a 96-well plate.

3. Add Bradford reagent to each well.

4. Incubate plate at room temperature for 5 min.

5. Measure the absorbance at 595 nm.

6. Subtract the blank and plot the standard curve.

7. Compare sample absorbances to the standard curve to determine protein concentrations of Cas.

 

After the transformants to be cultured were incubated for 16 h, the growing transformants were identified by colony PCR, and those with bands matching the expected size were the successful edited transformants. By comparing the knockdown efficiency of the bacteria using the new plasmid with the knockdown efficiency of the control group using the original plasmid, it can be known that the editing rate is improved.