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Experiments

Experiment
Protocol
Homologous Recombination
Protocol： 

  1. Calculate the amount of vector DNA and insert DNA fragments required using CE Design v1.04. Then calculate the amount of water required for a 20 μl system. 
  2. Mix the following agents in a 0.2 mL EP tube: 

Component 

Volume 

CE II Buffer (5×)

4 μl

Exnase II

2 μl

Vector DNA

Volume calculated by CE Design

Insert DNA

Volume calculated by CE Design

H2O

Volume calculated 

  1. Incubate at 37°C for 30 min.

Bacterial Transformation
Protocol: 

  1. Take chemically competent DH5α cells out of -80°C and immediately place it on ice. Extract 20 μL competent cells and add altogether 2 μL of desired plasmids (The volume of each plasmid solution added should be less than 10% of the total volume of the bacterial liquid, and the mass of each plasmid added should be greater than 100 ng). Gently flick the bottom of the EP tube for better mixing, then place the tube on ice for 30 min.
  2. Heat shock the cells in a 42°C water bath for 45 secs, then quickly put the tube back on ice and let it stand for 2 min.
  3. Add 700 μL of antibiotic-free LB liquid medium to the centrifuge tube. After mixing well, put the tube in the shaking incubator at 37℃ and resuscitate at 200 rpm for 60 min.
  4. Centrifuge the tube at 5000 rpm for 1 min and remove the upper supernatant, leaving approximately 100 μL liquid in the tube. Gently resuspend the bacteria in the media, and apply the mixture to LB agar plate containing antibiotic.
  5. Place the plate upside down in a 37℃ incubator for overnight culture.

Isolating Single Colonies from LB Plate
Protocol:

  1. Add 4 mL of antibiotic-free LB liquid medium to a sterile test tube and add 4 μL of corresponding antibiotic. Mix well.
  2. Pick a monoclonal population from the transformation plate using a sterile pipette tip and place it in the tube.
  3. Put the tube in the shaking incubator at 37°C for 12-14 h.

Plasmid Extraction (Performed according to AxyPrep Plasmid Miniprep Kit)
Reagent: 


RNase A

Store at room temperature for 6 months or -20°C for long-term storage.

Buffer S1

Resuspension buffer. Store at 4°C after addition of RNase A.

Buffer S2

Lysis buffer (containing SDS/NaOH). Store at room temperature.

Buffer S3

Neutralization buffer. Store at room temperature.

Buffer W1

Wash buffer. Store at room temperature.

Buffer W2 concentrate

Desalting buffer. Add ethyl alcohol before using. Store at room temperature.

Eluent

2.5 mM Tris-HCl, pH 8.5. Store at room temperature.

Preparation before experiment: 

  1. Before using, add the RNase A to Buffer S1. Mix and store at 4°C. 
  2. Add ethyl alcohol (Either 100% or 95%) to Buffer W2 before using.
  3. Check Buffer S2 for precipitation before using. If it occurs, dissolve the precipitate at 37°C and equilibrate to room temperature.

Protocol: 

  1. Collect 1-4 mL of overnight LB culture. Centrifuge at 12,000×g for 1 min. Decant the supernatant.
  2. Add 250 μl of Buffer S1. Resuspend the bacterial pellet. Be sure that the bacteria pellet is completely resuspended.
  3. Add 250 μl of Buffer S2 and gently inverting the tube for 4-6 times until a clear solution is formed. Buffer S3 must be added within 5 minutes. 
  • * The buffer S2 bottle should be closed immediately after using to avoid neutralization of NaOH by ambient CO2.* Avoid vigorous shaking or vortexing to prevent the contamination of the genomic DNA.
  1. Add 350 μl of Buffer S3, and gently inverting 6-8 times. Centrifuge at 12,000×g for 10 min. 

* Avoid vigorous shaking or vortexing to prevent contamination with genomic DNA. 

  1. Place a miniprep column into a 2 mL microfuge tube. Transfer the clarified supernatant from Step 4 to the miniprep column. Centrifuge at 12,000×g for 1 min. Discard filtrate from the 2 ml microfuge tube. 
  2. Place the miniprep column back into the 2 ml microfuge tube and add 500 μl of Buffer W1 into miniprep column. Centrifuge at 12,000×g for 1 minute. Discard filtrate from the 2 ml microfuge tube. 
  3. Place the miniprep column back into the 2 ml microfuge tube and add 700 μl of Buffer W2 into miniprep column. Centrifuge at 12,000×g for 1 minute. Discard filtrate from the 2 ml microfuge tube.
  4. Repeat step 7  

* Two washes with Buffer W2 ensure the complete removal of salt, eliminating potential problems in Enzyme digestion identification. 

  1. Discard filtrate from the 2 ml microfuge tube. Place the miniprep column back into the 2 ml microfuge tube. Centrifuge at 12,000×g for 1 minute. Discard filtrate from the 2 ml microfuge tube.
  2. Transfer the miniprep column to a 1.5 ml microfuge tube. To elute the purified plasmid DNA, add 60~80 μl of Eluent (or deionized water) to the center of the column. Let it stand for 1 min at room temperature. Centrifuge at 12,000×g for 1 min. 

  * Heat Eluent or deionized water to 65 °C to increase elution efficiency.
Colony PCR
Protocol: 

  1. Pick a single bacterial colony using a pipette tip and put the colony into 100 μl of LB liquid medium in a tube.
  2. Put the tube in a shaking incubator at 37°C for 1 h.
  3. Extract 1 μl of bacteria liquid as template for PCR.
  4. Combine the following in a system of 25μl using a PCR tube:

Component 

Volume

Template 

1 μl

Forward Primer 

0.75 μl

Reverse Primer 

0.75 μl

Green Taq Mix 

12.5 μl

H2O

10 μl

Place the tubes in a PCR amplifier and set the program as:


Step 

Temperature 

Time 

Denaturation 

95°C

10 min

Denaturation

95°C

15 secs

Annealing 

Varies according to the different annealing temperature of different primers 

15 secs

Extension 

72°C

Varies according to the length of the DNA segment (the speed of extension is 0.5-1 min/kb)

Repeat step 2-4 thirty times 

/

/

Final extension 

72°C

10 min

Hold 

12°C

∞

Strain Preservation
Protocol:

  1. Extract 300 μl of 30% glycerol into test tubes.
  2. Extract 300 μl of bacteria liquid into each of the test tubes.
  3. Store at -20°C for long term preservation.

SDS-PAGE (Performed according to Biochemistry Experiment and Guidance of China Pharmaceutical University)
Protocol:

  1. Mix the following reagents: (TEMED is added last)

Monomer running gel: 


Monomer running gel

6%

8%

10%

12%

15%

dH20

5.3 ml

4.6 ml

4.0 ml

3.3 ml

2.3 ml

30%Acrylamide

2.0 ml

2.7 ml

3.3 ml

4.0 ml

5.0 ml

1.5M Tris-base pH8.8

2.5 ml

2.5 ml

2.5 ml

2.5 ml

2.5 ml

10%SDS

0.1 ml

0.1 ml

0.1 ml

0.1 ml

0.1 ml

10%APS

0.1 ml

0.1 ml

0.1 ml

0.1 ml

0.1 ml

TEMED

0.008 ml

0.006 ml

0.004 ml

0.004 ml

0.004 ml

5% monomer stacking gel: 


Monomer stacking gel

5%

dH20

3.4 ml

30% Acryclamide

0.83 ml

1.0M Tris-base PH6.8

0.63 ml

10% SDS

0.05 ml

10% APS

0.05 ml

TEMED

0.005 ml

  1. After the glass plates are aligned, put them into the rubber frame and clamp them (the plates must be aligned to avoid leakage). Then clamp the clip.
  2. Use pipette to extract 1 mL of running gel solution and release it into spaces between parallel glass, until it reaches 5 mm below the line. Create a flat surface above the running gel solution by adding a water layer on top of the solution. Allow the gel to polymerize for at least 30 min.
  3. When there is a refracting line between running gel and water layer, pour off the water. Wash the gel for several times with deionized water and blot the water with filter paper.
  4. Pour the stacking gel above the running gel.
  5. Immediately insert the comb to create sample wells in the stacking gel before it polymerizes (keep the comb level). Be sure that the comb is clean and that no air bubbles form. Allow the gel to polymerize for at least 30 min.
  6. Place the gel in electrophoresis tank with small glass plate facing inward. Add running buffer to the chambers at the bottom and top of the gel. Gently remove the comb (The running buffer in the upper tank should pass through the small glass plate edge, and the running buffer in the lower tank should pass through the wire). Be sure that no bubbles interfere with uniform contact of the liquid in the running buffer with the gel at the bottom or at the top.
  7. Use running buffer to wash stacking gel. Remove the redundant acrylamideleft by the comb to avoid deformation of the strip).
  8. After concentration testing, calculate the volume containing 20-50 μg protein as sample. Transfer sample to 200 μl EP tube and add 5x protein buffer until the buffer concentration reaches 1x. The loading volume should be 15 μl (10-20 μl) and the Marker volume should be 3μl on the left and 1μ on the right for small gel and 4μl on the left and 2μ on the right for large gel.
  9. Connect the electrodes of the apparatus to the power source. The anode (+ pole) must be at the bottom of the apparatus. Apply a constant 80V field to the apparatus for 30 min when running the stacking gel and apply a constant 120V field to the apparatus for 60 min when running the running gel. Stop electrophoresis when Bromophenol Blue tracking dye reaches the very bottom of the gel.
  10. Staining and destaining:

Method I:

  1. Mix following reagents;

Coomassie Brilliant Blue (100 ml in total): 


Component 

Volume

Methanol

45 ml

H2O

45 ml

Glacial acetic acid

10 ml

R-250

0.25 g

  1. Gently transfer the running gel to a pan filled with approximately 50 ml of Coomassie Blue staining solution and allow it to soak in the solution for 1 h.
  2. Mix following reagents;

Destaining solution (2000 ml in total):


Component 

Volume

Methanol

900 ml

H2O

900 ml

Glacial acetic acid

200 ml

  1. Pour off the staining solution and replace it with destaining solution for 1 hour. Replace the gel with fresh destaining solution and gently agitate the gel in the solution continuously during destaining to shorten the destaining time. Destaining is complete when the proteins appear as blue bands on a transparent gel background.

Method II: (Performed according to https://www.genscript.com/eStain-L1-protein-staining-system.html)
Use eStain® L1 Protein Staining System to stain and destain gels.

  1. Add distilled water into the tray provided. When gel electrophoresis is complete, carefully remove the gel from the gel cassette and briefly rinse the gel with distilled water for 1 min;
  2. Open the gel holder and place it on the table with the fabric side down. Carefully place the rinsed gel on the fabric side of the gel holder. Be sure that the upper part of the gel (with comb) point to the axis of the gel holder and is as close to the axis as possible;
  3. Pre-wet a piece of filter paper (provided) with distilled water and place it on top of the gel;
  4. Close the gel holder;
  5. Insert the gel holder into the channel;  

* Be sure that the fabric side of the gel holder is facing the user before inserting it into the Channel.

  1. Press the corresponding channel to start the program. The instrument beeps as the program countdown to 0;
  2. Take out the gel holder and the gel.

PCR (Performed according to Vazyme Mut Express II Fast Mutagenesis Kit V2) 

  1. Combine the following in a PCR tube:
    1. For a system of 25 μl:

Component 

Volume

Template 

1 μl

Forward Primer 

0.75 μl

Reverse Primer 

0.75 μl

Prime Star (2×)

12.5 μl

H2O

10 μl

  1. For a system of 50 μl:

Component 

Volume

Template 

2 μl

Forward Primer 

1.5 μl

Reverse Primer 

1.5 μl

Prime Star (2×)

25 μl

H2O

20 μl

  1. Put the PCR tube into a PCR amplifier and set the program as:

Step

Temperature

Time

1. Denaturation

95°C

2 min

2. Denaturation

95°C

15 sec

3. Annealing

Varies according to the different annealing temperature of different primers.

15 sec

4. Extension

72°C

Varies according to the length of the DNA segment (the speed of extension is 0.5-1min/kb)

5. Repeat step 2-4 thirty times

/

/

6. Final extension

72°C

10 min

7. Hold

10°C

∞

Agarose Gel Electrophoresis

  1. Add 0.25 g of Agarose B, Low EEO (Sangon Biotech Co., Ltd., Shanghai) to the flask.
  2. Add 25 mL of 1× TAE.
  3. Heat in a microwave oven until the solution clarifies.
  4. Cool down the solution at room temperature until hands can be comfortably kept on the flask.
  5. Add 1 μL of Ultra GelRed (Vazyme Biotech Co.,Lt., Nanjing). Shake gently to mix well.
  6. Pour the agarose solution into a gel tray with the well comb in place and wait for the gel to solidify.
  7. Once solidified, take out the comb and place the agarose gel into the gel box. Be sure that the sample wells are placed toward the negative electrode. Fill the gel box with 1xTAE until it covers the surface of the gel.
  8. Add 2.8 μL of 10x Loading Buffer to each sample (25 μL) and mix well.
  9. Add 2 μL of Marker into the first well.
  10. Carefully load 27.8 (2.8+25) μL of each of the samples into corresponding wells of the gel.
  11. Put on the cover, plug in the power supply, and set the time and voltage at desired value. Be sure that a row of bubbles is observed from the negative electrode.
  12. Turn OFF power after desired time, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box. Using device that has UV light to visualize the DNA segment. Take a photo and record the results.

DNA gel extraction (Performed according to AxyPrep DNA Gel Extraction Kit)
Reagent: 


Buffer DE-A

Gel solubilization buffer with DNA protective agent to avoid degradation at high temperature. Store at room temperature.

Buffer DE-B

Binding buffer. Store at room temperature.

Buffer W1

Wash buffer. Store at room temperature.

Buffer W2 concentrate

Desalting buffer. Add ethanol before using. Store at room temperature.

Eluent

2.5 mM Tris-HCl, pH 8.5. Store at room temperature.

Caution：

  1. In step 1, cutting the gel into small pieces can increase extraction rate by greatly shortening the gel dissolving time. Do not expose DNA-containing gels to UV light for long periods of time to reduce UV damage to DNA.
  2. In step 2, the gel must be completely dissolved, otherwise the extraction rate will be severely affected.
  3. Improve elution efficiency by pre-warming Eluent to 65°C.
  4. The DNA molecule is acidic. Recommended to store the DNA in 2.5 mM Tris-HCl, pH 8.5 Eluent.

Preparation before experiment:

  1. Before using, add ethyl alcohol specified on the bottle label to the Buffer W2 concentrate. 
  2. Make sure pipette tip and Microfuge tube are without nucleic acid and nuclease contamination. 
  3. Adjust water bath to 75°C. 
  4. Check Buffer S2 for precipitation before each use. If precipitation occurs, incubate at 70°C to dissolve the precipitate and then equilibrate to room temperature.

Protocols:

  1. Excise the DNA-containing agarose gel slice with a clean, sharp scalpel under ultraviolet illumination. Briefly place the excised gel slice on absorbent toweling to remove redundant buffer. Mince the gel into small pieces, transfer to a 1.5 ml microfuge tube, and weigh (Record the weight of the 1.5 ml centrifuge tube in advance). In this application, the weight of gel is regarded as equivalent to the volume. For example, 100 mg of gel is equivalent to a 100 μl volume;
  2. Add a 3x sample volume of Buffer DE-A. Resuspend the gel in Buffer DE-A by vortexing and heat at 75°C. Intermittently vortexing (every 2-3 minutes) until the gel is completely dissolved (typically, 6-8 min later);
  3. Add 0.5x Buffer DE-A volume of Buffer DE-B, mix.
  4. Place a miniprep column into an uncapped 2 ml microfuge tube and transfer the binding mix from Step 3 to the miniprep column. Centrifuge at 12,000×g for 1 minute. Discard filtrate from the 2 ml microfuge tube.
  5. Place the miniprep column back into the 2ml microfuge tube and add 500 μl of Buffer W1 into miniprep column. Centrifuge at 12,000×g for 30 s. Discard filtrate from the 2 ml microfuge tube.
  6. Place the miniprep column back into the 2ml microfuge tube and add 700 μl of Buffer W2 into miniprep column. Centrifuge at 12,000×g for 30 s. Discard filtrate from the 2 ml microfuge tube. Place the miniprep column back into the 2 ml microfuge tube and add 700 μl of Buffer W2 to the miniprep column again. Centrifuge at 12,000×g for 30 s.  
  • * Be sure that the ethyl alcohol is added to Buffer W2 concerate.  
  • * Two washes with Buffer W2 ensure the complete removal of salt, eliminating potential problems in subsequent Enzyme digestion identification. 
  1. Discard filtrate from the 2 ml microfuge tube. Place the miniprep column back into the 2 ml microfuge tube. Centrifuge at 12,000×g for 1 minute. Discard filtrate from the 2 ml microfuge tube.
  2. Transfer the miniprep column into a 1.5 ml microfuge tube. To elute the purified plasmid DNA, add 60~80 μl of Eluent (or deionized water) to the center of the column. Let it stand for 1 min at room temperature. Centrifuge at 12,000×g for 1 minute.  

* Heat Eluent or deionized water to 65 °C to increase elution efficiency.
Measuring the concentration of nucleic acid
Equipment used: 
Nano-300 Micro Spectrophotometer, Hangzhou Allsheng Instruments CO., Ltd., Hangzhou, China
Protocol:

  1. Pipette 2 μL of water and drip it onto the sample pedestal. Close the micro spectrophotometer, reopen it, put Kimtech wiper on the sample plate, and close it to clean the plate;
  2. Select nucleic acid detection mode on the screen;
  3. Pipette 2 μL of Eluent solution and drip it onto the sample pedestal. Close the micro-spectrophotometer and select Blank Calibration on the screen;
  4. Pipette 2 μL of sample and drip it onto the sample pedestal. Be sure that the droplet is on the pedestal. Close the micro spectrophotometer, and select Sample Measurement;
  5. Record data;
  6. To clean the sample pedestal, open the micro spectrophotometer, place the wiper on the sample pedestal, close the micro spectrophotometer, and open it again. Repeat the steps for 1-2 times.
  7. Repeat steps 4-6 until all samples are measured;
  8. Repeat step 1 after using.

Electrotransformation
Preparation for electrocompetent cells:

  1. Take 10 μL of bacteria liquid and put it into 3 mL of LB medium;
  2. Incubate the bacteria at 37 ° C, 220 rpm in shaking incubator for 12-16 h.
  3. Mix the bacteria with 100 mL of LB medium at the ratio of 1:100. Incubate at 37 ° C, 220 rpm for 4-5 h until OD600=0.4~0.6.
  4. Pre-cool the microfuge tube (50 mL, 1.5 mL) and 10% glycerol, and prepare liquid nitrogen.
  5. Transfer the mixture of bacteria with OD600=0.4~0.6 to the pre-cooled 50 mL microfuge tube. Centrifuge the mixture at 6000 rpm for 15 min at 4 °C.
  6. Discard the supernatant. Add 10% glycerol (for 100 mL of cell mixture, 60 mL of glycerol shall be added), and resuspend the bacterial pellet. Centrifuge at 6000 rpm for 15 min at 4 °C.
  7. Repeat step 6 for once.
  8. Discard the supernatant and add appropriate amount of 10% glycerol. Pack 100 μL of the mixture into each tube, and immediately place the tubes in liquid nitrogen. Transfer to -80 °C for storage.

Electroporation: 

  1. Put the electroporation cuvette into laminar airflow bench. Turn on the UV light to sterilize the cuvette. Pre-cool the cuvette on ice for 30 min.
  2. Place 50 μl of electro competent cells in the labeled cuvette on ice.
  3. Add 5 µL of plasmid to electro competent cell, mix gently, and let it stand on ice for 2 min.
  4. Transfer the mixture into pre-cooled cuvette. Put the cuvette into the cuvette chamber of the electroporator. Electroporate under the condition of 2.5 kv/cm, 200 Ω, 25 µF for 5 ms Add 500 µL of pre-cooled LB medium to the cuvette immediately after electric shock.
  5. Transfer the mixture to Microfuge tube. Shake and culture at 180 rpm and 37℃ for 1 h.
  6. Centrifuged at 5000 rpm for 5 min. Discard the supernatant.
  7. Mix the cells and transfer to LB plates.

Experiment
To build the Light-Catch system, we divided ourteam into three groups:
Group one: BL21-AI cells with pSB1C3-fixk2-Cas1Cas2 and pCDF-T7-Blue Sensor.
The fist group constructed these cells by transforming pSB1C3-FixK2-Cas1Cas2 and pCDF-T7-Blue Sensor to BL21-AI cells.
We first transformed Blue Sensor and pSB1C3 Vector to BL21-AI. Then we used PCR to construct pSB1C3-fixk2-Cas1Cas2 and pCDF-J23100-Blue Sensor. We changed the promoter on pCDF-J23100-Blue Sensor with T7 to construct pCDF-T7-Blue Sensor.
Group two: BL21-AI cells with pET28a-fixk2-TfusCas3, pCDF-T7-Cas1Cas2, and pACYC-J23100-Blue Sensor:
The second group constructed these cells by transforming pET28a-fixk2-TfusCas3, pCDF-T7-Cas1Cas2, and pACYC-J23100-Blue Sensor to BL21-AI cells.
We first transformed pSB1C3-FixK2-Cas1Cas2 and Blue Sensor to BL21-AI. Then we used PCR to construct pFixK2-Cas3 Gene and pFixK2-Cas3 Vector, which were then transformed into DH5α and used to construct pET28a-FixK2-Cas3. Because we have obtained pSB1C3-FixK2-Cas1Cas2 and pCDF-T7-Blue Sensor in the experiments done by group one, we first changed the promoter on pSB1C3-FixK2-Cas1Cas2 and pCDF-T7-Blue Sensor with T7 and J23100, and then we used restrictive enzyme to cut the target gene and performed ligation to construct pCDF-T7-Cas1Cas2, and pACYC-J23100-Blue Sensor.
*Group three: BL21-AI cells with J23100-YF1-FixJ and pSB1C3-FixK2-GFP:
The third group constructed these cells by transforming J23100-YF1-FixJ and pSB1C3-FixK2-GFP to BL21-AI cells.
Unfortunately, because we failed to obtain pSB1C3-FixK2-GFP for several times, we decided to give up this experiment and focused on first two experiments introduced above.