The procedure is based on the Trans5a conversion manual, with minor modifications, and is as follows.

1 10 μL of ligation product was added to 50 μL of just melted competent cells, mixed gently, and left on ice for 30min.

2. Heat shock at 42° C for 45 s, quickly transfer to an ice bath and place for 2min. Please do not shake the centrifuge tube during this process.

3. Add 500μL LB liquid medium（Kan+）, 37℃, 200 rpm, and incubate for 1h to revive the bacteria.

4, Centrifuge the bacteria at 5000 rpm for 2 minutes. Collect the bacteria and discard a portion of the supernatant. Resuspend the remaining bacteria and inoculate them onto an LB liquid medium（Kan+） Incubate the culture overnight (12-14h) in an inverted incubator at a constant temperature of 37℃.

The methodology follows Han (2016) with slight modifications as follows:

The recombinant plasmid pET-Cas12a was transferred into the expression strain BL21 sensory state and, after the correct identification of colonies, prokaryotic expression was carried out.

1. BL21 colonies containing the correctly identified recombinant plasmid pET-Cas12a were inoculated into 5 mL LB liquid medium（Kan+）at 37 ℃, 200 rpm, for shock culture for 12-16 h.

2. Absorb 2 mL of the bacterial solution cultured overnight into 200 mL of LB liquid medium（Kan+）, and culture with shaking at 37 ℃, 200 rpm, until the bacterial solution is about OD600 to 0.5, about 2-3 h.

3. 1.5 mL bacterial solution was aspirated as a whole cell control before IPTG induction and stored at -20 ° C

4. Add IPTG until the final concentration is 0.5mmol /L, 1mmol /L, and 1.5mmol /L, respectively, at 37 ℃ and 150 rpm, and oscillate for 3-6 h.

5. 1.5 mL of bacterial solution was aspirated as a whole cell control after IPTG induction and stored at -20 ° C.

6, The rest of the bacteria were centrifuged at 5000 rpm and 4 ° C for 5 min, and the bacteria were collected. After the supernatant was discarded, the bacteria were repeatedly frozen and thawed 3 to 4 times with liquid nitrogen (if liquid nitrogen was not available, the bacteria were washed twice with PBS, resuspended with 1ml lysate, and added with a final concentration of 1mmol/L PMSF solution. Crushed on ice with an ultrasonic crusher).

7. The bacteria were resuspended in 20 mL of ice-cold PBS for precipitation, and then crushed by an ultrasonic crusher on ice until the bacterial solution was translucent to avoid bubbles, and ultrasonic crushing was carried out on ice.

8. After centrifugation at 10,000 rpm at 4 ℃ for 10 min, the supernatant and precipitate were aspirated and subjected to SDS-PAGE to observe the expression and dissolution of GST fusion protein. If the expression of the pET-Cas12a fusion protein is high and in the supernatant, proceed to the next purification test. If the expression of the pET-Cas12a fusion protein is low or in precipitation, IPTG concentration and incubation temperature should be optimized.

1. Prepare LB liquid medium（Kan+）500 ml to culture Cas12a E. coli, weigh the following reagents with an electronic balance, and add them into 4 conical flasks using a spatula.Tryptone* 5 g

*Tryptone is used to provide essential amino acids such as peptides and peptones to the bacteria

**Yeast Extract provides a range of organic compounds for bacteria growth

***Sodium chloride balances osmotic pressure and provides sodium ions for the bacterium

****ddH2O: double distilled water

2.Autoclave all LB (lysogeny broth)

*No need to adjust the pH as both sodium hydroxide and TRIS buffer are usually unnecessary.

3. Allocate 150ml autoclaved LB into two equal aliquots, each 75ml.

4. Add 75μl of Kan+ antibiotics into one flask, and 75μl of Amp+ antibiotics into the other (as the volume ratio between LB and antibiotics should be 1000:1). Label the flasks.

5. Prepare 4 sets of cell culture tubes, each with 3 tubes.

6. In each group of tubes, allocate 5ml of Amp+ LB into one tube，and 5ml Kan+ LB into the other two.

7. Inoculate bacterium (Add 50μl of E. coli-containing-medium into each tube, ending up with four tubes with four different types of GM E. coli), and label the date, its antibiotics, and integrated sequence on the tube. The number and types of cell culture tubes are recorded in the table below:

8. Place all tubes in the shaker rock for 16 hours, and allow it to proliferate.

Prepare pulse buffer

Tris-glycine-sds buffer instant pellet was added to 1000ml of deionized water.

Dispensing gel

1. 2.5ml separation gel solution A and 2.5ml separation gel solution B were mixed

2. Add 25μl coagulant polymerization agent to mix, quickly inject the mixture into the gelatin glass plate (0.5-1cm comb tooth)

3. Insert the comb teeth and wait for solidification

Concentrate gel

10μl of gel polymerizer was mixed, and added to the rubber plate.

Whole Protein Extraction

1. Add 10µl of phosphate inhibitor and 10µl of protease inhibitor PMSF to 1ml of cold lysate, mix well and place on ice.

2. Take 1-5mL of the bacterial solution and centrifuge at 12000rpm for 1 minute, discard the supernatant.

3. Add 0.5-1ml of the new solution to the bacterial block and vortex until well mixed.

4. Transfer the liquid to a 1.5ml EP tube and centrifuge at 4℃, 12000 rpm for 30 minutes.

5. Aspirate the supernatant into a new tube (store the protein at -80°C).

6. Protein gel electrophoresis (300v,20min) was performed.

Collect E. coli bacteria

Purpose: to separate it from LB Centrifugation the bacteria medium at 4000 rpm

Discard the supernatant

1.Suspend E. coli within Buffer A

2.Add 5 ml of Buffer A into the sediments, allow the biomass to float

Ultrasound Cell Lysis of E. coli

Purpose: Break the bacteria membranes so that Cas12a proteins can be extracted and purified

1.Place the entire sample on ice (maintain a temperature of 4 °C to preserve its proteins) and place it into an ultrasound cell crusher.

2.Set the total time as 10 minutes and the interval as 5 seconds (to close for 5 seconds every time the system worked for 5 seconds). The power ratio should be at 70%.

Centrifugation of Ultrasound Results.

Place the sample in the centrifuge. Set the system at 4000 rpm, 20 minutes, 4°C.

After centrifugation, extract the supernatant (containing CAS 12a proteins)

Nickel affinity purification of Cas12a

Principle: The 6×His tail of the Cas12a protein is strongly attracted to the nickel column and is therefore not washed away by buffer A, but is washed away by buffer B. Whereas other proteins, which adhere more or less strongly than Cas12a, are either washed away by the addition of buffer A or not washed away by buffer B.

1. Mix the reagents below in the assigned portion as shown in the chart to obtain 0.5 L His buffer A

2. Mix the reagents below in the correct portion as mentioned in the chart below and obtain 0.25 His buffer B

1. Complete installing the nickel affinity purification system by stacking the nickel column onto a new microtube. Then, place it on ice.

2. Using a pipette, add 4 times the nickel column’s size worth of Buffer A to moist the column. Collect one tube of the solution under the nickel column and label it as the “first filter”.

3. Add the supernatant into the nickel column. Decant slowly and gradually. Keep the velocity of the flow at 1ml/min.

4. Add 6 ml of Buffer A into the nickel column. Keep the velocity of the flow at 1ml/min.

5. Collect one tube of the solution under the nickel column and label it as the“last filter”.

6. Repeat step 4 for two more times.

7. Add 3 ml of Buffer B into the nickel column. Keep the velocity of the flow at 1ml/min.

8. Collect two tubes of the solution under the nickel column and label them as “Cas12a protein #1” and “Cas12a protein #2” respectively. They are the target protein.

9. Add 2 ml of His Buffer B into the nickel column.

10. Collect one tube of the solution under the nickel column and label it as “Buffer B elution”.

11. Add 5 ml of Buffer A into the nickel column.

12. Collect one tube of the solution under the nickel column and label it as “Buffer A elution”.

13. Wash the nickel column by decanting 10ml of His buffer A and 5ml 20% ethanol. Measure the concentration of Cas12a protein in the resultant solution

1、Wash off any residue on the NanoDrop using ddH2O.

2、Add 2μl of Buffer B onto the probe using a pipette (As the negative control group). Record the protein concentration. Wash off any residue on the NanoDrop.

3、Add 2μl of “Cas12a protein #1” solution onto the probe using a pipette. Record the protein concentration. Wash off any residue on the NanoDrop.

4、Add 2μl of “Cas12a protein #2” solution onto the probe using a pipette. Record the protein concentration. Wash off any residue on the NanoDrop.