6.1 Transcription of Cas12a proteins in E. coli
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
- Prepare 3 conical flasks, each holding 150ml of LB medium
- Add 150μl of antibiotic K+ into each flask using a pipette (note: add after the K+ solution fully
melts, as it was previously frozen)
- 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)
- 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
- Wipe off any residues on the Nanodrop from previous users with lens paper
- Add 1μl of LB onto the probe using a pipette as the control group.
- 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
- Add 30μl of IPTG into flask 1 (containing 150ml LB and E. coli), creating a concentration of
0.2μM/μl
- Add 75μl of IPTG into flask 2 (containing 150ml LB and E. coli), creating a concentration of
0.5μM/μl
- Add 120μl of IPTG into flask 3 (containing 150ml LB and E. coli), creating a concentration of
0.8μM/μl
6.2. Cas12a protein extraction
6.2.1. Purify Cas12a Protein
-
Collect E. coli bacteria
Purpose: to separate it from LB
- Centrifugation the bacteria medium at 4000 rpm
- Discard the supernatant
-
Suspend E. coli within Buffer A
- 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
- Place the entire sample on ice (maintain a temperature of 4 °C to preserve its proteins) and
place it into an ultrasound cell crusher.
- 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)
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.
-
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 |
-
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 |
- Complete installing the nickel affinity purification system by stacking the nickel column onto a
new microtube. Then, place it on ice.
- 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”.
- Add the supernatant into the nickel column. Decant slowly and gradually. Keep the velocity of
the flow at 1ml/min.
- Add 6 ml of Buffer A into the nickel column. Keep the velocity of the flow at 1ml/min.
- Collect one tube of the solution under the nickel column and label it as the“last filter”.
- Repeat step 4 for two more times.
- Add 3 ml of Buffer B into the nickel column. Keep the velocity of the flow at 1ml/min.
- 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.
- Add 2 ml of His Buffer B into the nickel column.
- Collect one tube of the solution under the nickel column and label it as “Buffer B elution”.
- Add 5 ml of Buffer A into the nickel column.
- Collect one tube of the solution under the nickel column and label it as “Buffer A elution”.
- 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
- Wash off any residue on the NanoDrop using ddH2O.
- 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.
- Add 2μl of “Cas12a protein #1” solution onto the probe using a pipette. Record the protein
concentration. Wash off any residue on the NanoDrop.
- Add 2μl of “Cas12a protein #2” solution onto the probe using a pipette. Record the protein
concentration. Wash off any residue on the NanoDrop.
6.3. Protein electrophoresis of Cas12a proteins
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)
- In a sterile cup, add 2ml of 2× gel solution A and 2ml of gel solution B
- Using a pipette add 55μl TEMED, and gently swirl the cup until all reagents are fully mixed
- Add the mixture gently, aware not to introduce any gas bubbles
- Add ethanol using a pipette until the liquid level reaches the top
- Wait for 6 to 10 minutes at room temperature, and allow the separating gel to solidify. After the
gel solidifies, dispose of the ethanol.
- 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
- Gently pour the mixture between two glass plates (mold) and insert a comb on the top
- Wait for 10-15 minutes until the stacking gel solidify.
- Extract the comb.
6.3.2. Protein electrophoresis of Cas12a proteins
-
Use a pipette to inject 40μl of protein into each of the seven microtubes in the order of nickel
column filter.
- first filtrate
- last filtrate
- Cas12a1
- Cas12a2
- Buffer B
- Buffer A
- Bacteria sediment (obtained from the residue in “purifying Cas12a proteins”)
- Add 10μl of 4× loading buffer into each of the seven microtubes
-
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.
-
Make Tris-Glycine SDS buffer by mixing the following reagents.
- 1 pack of Tris-Glycine SDS buffer powder
- 1L ddH2O
- 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.
- Make sure the positive and the negative electrodes are plugged in correctly.
- Add 20μl of the sample protein (7 samples in total) into separate tubes carefully.
- Add three markers at 20μl, to fill in all wells and ensure each tube has the same weight.
- Start the electrolysis with a setting of 180V, for 45 minutes.
- When the machine is turned on, air bubbles should travel upwards (opposite from the electrical
current).
- 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.
- Put the dyed protein gel onto the horizontal oscillator and incubate for 90 min.
- Wash the protein gel using Coomassie brilliant blue decolorizing solution and soak it in the same
solution for 18 hours
- Place gel into GenoSens 2000 Gel Documentation and Analysis System to collect results.
6.3.3. BCA test of Cas12a proteins’ concentration
- Dilute 20μl 5mg/ml BSA standard solution with 250μl DD water into a beaker to produce 0.04μg/ml BSA
standard protein solution
- In 9 of the wells on the lightproof microplate, add 100μl BCA reagent and 100μl standard protein
solution
- In 2 of the wells on the lightproof microplate, add 100μl BCA reagent and 100μl sample Cas12a
protein
solution
- Incubate the reagents in a gradient thermal cycler at 37 °C for 30 minutes.
- Test using Subtract max