6.1.1 Transfer cas12a-containing E. coli for reproduction at a greater scale

Purpose: For greater amount of E. coli cloning
Reagents: LB, antibiotic K+, Cas12a containing E. coli
  1. Prepare 3 conical flasks, each holding 150ml of LB medium
  2. Add 150μl of antibiotic K+ into each flask using a pipette (note: add after the K+ solution fully melts, as it was previously frozen)
  3. Add 5μl bacterial solution that contains Cas12a into each flask (while the normal ratio of E. coli to LB is 1:1000, we increased the ratio to accelerate the speed of bacterial cloning at a greater scale)
  4. Place the flasks into a shaker and incubate for 5 hours

6.1.2. Measure OD value of E. coli (dedicated to inducing Cas12a protein expression)

Purpose: Check if the concentration is suitable for the addition of IPTG
Reagents: LB, antibiotic K+, Cas12a containing E. coli
  1. Wipe off any residues on the Nanodrop from previous users with lens paper
  2. Add 1μl of LB onto the probe using a pipette as the control group.
  3. Run the test (by selecting the “nucleic acid” option)

6.1.3. Addition of IPTG

Purpose: Promote the transcription of Cas12a proteins in E. coli colonies in different IPTG environments while also allowing comparison
Reagents: LB, IPTG
  1. Add 30μl of IPTG into flask 1 (containing 150ml LB and E. coli), creating a concentration of 0.2μM/μl
  2. Add 75μl of IPTG into flask 2 (containing 150ml LB and E. coli), creating a concentration of 0.5μM/μl
  3. Add 120μl of IPTG into flask 3 (containing 150ml LB and E. coli), creating a concentration of 0.8μM/μl

6.2.1. Purify Cas12a Protein

  1. Collect E. coli bacteria
    Purpose: to separate it from LB
    1. Centrifugation the bacteria medium at 4000 rpm
    2. Discard the supernatant
  2. Suspend E. coli within Buffer A
    1. Add 5 ml of Buffer A into the sediments, allow the biomass to float
  3. 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%.
  4. Centrifugation of Ultrasound Results
    1. Place the sample in the centrifuge. Set the system at 4000 rpm, 20 minutes, 4 °C.
    2. After centrifugation, extract the supernatant (containing CAS 12a proteins)

6.2.2. Nickel affinity purification of Cas12a protein

Principle: The 6×His tail of Cas12a protein is strongly attracted to the nickel column, therefore will not be washed down by Buffer A but will be washed down by Buffer B. Whereas other proteins, attach more firmly or less firmly than the Cas12a, thus will be cleaned by adding Buffer A or will not be washed 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
    Buffer A: 0.5L
    Ingredients
    20 mM Na2HPO4.2H2O 1.78 g
    500 mM NaCl 14.6 g
    20 mM Imidazole 1.02 g
    HCl (6M) until pH reaches 7.4 and the volume reaches 0.5L
  2. Mix the reagents below in the correct portion as mentioned in the chart below and obtain 0.25 His buffer B
    Buffer B: 0.25L
    Ingredient
    20 mM Na2HPO4.2H2O 1.89 g
    500 mM NaCl 7.3 g
    500 mM Imidazole 8.5 g
    HCl (6M) until pH reaches 7.4 and the volume reaches 0.25L
  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

6.2.3. 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.
Purpose: To test the content and purity of the extracted Cas12a proteins
Material: Protein 40μl, 4× Loading buffer 10μl, 7 microtubes

6.3.1 Protein Electrophoresis preparation (1mm thick mm gel)

  1. In a sterile cup, add 2ml of 2× gel solution A and 2ml of gel solution B
  2. Using a pipette add 55μl TEMED, and gently swirl the cup until all reagents are fully mixed
  3. Add the mixture gently, aware not to introduce any gas bubbles
  4. Add ethanol using a pipette until the liquid level reaches the top
  5. Wait for 6 to 10 minutes at room temperature, and allow the separating gel to solidify. After the gel solidifies, dispose of the ethanol.
  6. Mix 0.75ml 2× upper gel solution A and 0.75ml 2× colored upper gel solution B (red) and 15μl TEMED in a sterile cup, gently swirl the cup
  7. Gently pour the mixture between two glass plates (mold) and insert a comb on the top
  8. Wait for 10-15 minutes until the stacking gel solidify.
  9. Extract the comb.

6.3.2. Protein electrophoresis of Cas12a proteins

  1. Use a pipette to inject 40μl of protein into each of the seven microtubes in the order of nickel column filter.
    1. first filtrate
    2. last filtrate
    3. Cas12a1
    4. Cas12a2
    5. Buffer B
    6. Buffer A
    7. Bacteria sediment (obtained from the residue in “purifying Cas12a proteins”)
  2. Add 10μl of 4× loading buffer into each of the seven microtubes
  3. Then put the tubes into a 100℃ metal bath for 10 minutes. This is to break the bonds in proteins and disrupt their complex dimensional structures so that they will travel in a regular route during the electrophoresis.
  4. Make Tris-Glycine SDS buffer by mixing the following reagents.
    • 1 pack of Tris-Glycine SDS buffer powder
    • 1L ddH2O
  5. Fill the middle cell in the protein electrophoresis machine with Tris-Glycine SDS buffer, and let it evenly overflow into the two cells on the side. Make sure the water level in the outer cells exceeds half of the container’s size.
  6. Make sure the positive and the negative electrodes are plugged in correctly.
  7. Add 20μl of the sample protein (7 samples in total) into separate tubes carefully.
  8. Add three markers at 20μl, to fill in all wells and ensure each tube has the same weight.
  9. Start the electrolysis with a setting of 180V, for 45 minutes.
  10. When the machine is turned on, air bubbles should travel upwards (opposite from the electrical current).
  11. After the electrolysis, use Coomassie brilliant blue to stain the protein gel (that contains the seven samples), which then visualizes the protein traces after destaining.
  12. Put the dyed protein gel onto the horizontal oscillator and incubate for 90 min.
  13. Wash the protein gel using Coomassie brilliant blue decolorizing solution and soak it in the same solution for 18 hours
  14. Place gel into GenoSens 2000 Gel Documentation and Analysis System to collect results.

6.3.3. BCA test of Cas12a proteins’ concentration

  1. Dilute 20μl 5mg/ml BSA standard solution with 250μl DD water into a beaker to produce 0.04μg/ml BSA standard protein solution
  2. In 9 of the wells on the lightproof microplate, add 100μl BCA reagent and 100μl standard protein solution
  3. In 2 of the wells on the lightproof microplate, add 100μl BCA reagent and 100μl sample Cas12a protein solution
  4. Incubate the reagents in a gradient thermal cycler at 37 °C for 30 minutes.
  5. Test using Subtract max