Team:NPU-CHINA/Protocols
PROTOCOL &
LAB WORK
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The homologous recombination cloning scheme we use
This method has the highest success rate among the methods we have tried.
1. The primer design follow the seamless cloning principles, and it is recommended to use software such as snapgene to design. When designing primers, we follow the following points: ①: Control the length of the overleg at 20-25bp ②: Control the Tm of the homologous fragment at 50-60℃. If you use the Gibson assembly in snapgene to design primers, generally set the Tm value to 55°C or 60°C.
2. Design the reverse of the plasmid according to the primer design scheme, and obtain the linearized plasmid by PCR technology. Note: High-fidelity PCR enzymes must be used when performing PCR on plasmids, and the reaction solution is recommended to be no less than 50μL.
PCR program:①: 94℃ 5min
②: 98℃ 10sec
③: 55℃ 20sec
④: 72℃ 1000bp/min
Goto② for 30 cycles
⑤: 72℃ 10min
3. Design the primers of the gene fragments according to the primer design scheme, and obtain the target gene fragments by PCR technology. Note: It is recommended to use high-fidelity PCR enzymes when performing PCR, and the reaction system is recommended to be no less than 50μL. PCR program:
①: 94℃ 3min
②: 98℃ 10sec
③: 55℃ 20sec
④: 72℃ 1000bp/min or 1min30sec
Goto② for 30 cycles
⑤: 72℃ 5min
4. After the linear plasmid PCR is over, add 1μL of DpnI enzyme or DMT enzyme to the reaction solution to digest the template and reduce the probability of false positives in the subsequent process. Place at 37°C to react for 1 hour.
5. Separate and purify the target gene and linearized plasmid by electrophoresis and gel extraction. Note:
①: Try to use a newer gel extraction kit to ensure the extraction rate, and it should be carried out in strict accordance with the operating protocols, especially the process time of the gel solubilization not be too long.
②: The final concentration should not be less than 10ng/μL.
③: Nucleic acid dyes that are suitable for gel extraction should be selected when performing electrophoresis.
6. We use vazyme® ClonExpress II kit for homologous recombination cloning. The reaction system is as follows:
Reagent Volume Exnase 2μL 5x CE buffer 4μL Vector Vector length（bp）*0.02/concentration of vector Insert fragement fragement length（bp）*0.04/concentration of fragement ddH2O Up to 20 μL React at 37°C for 30 minutes.
Note:
①: The actual amount of plasmid and gene fragment added should not be less than 1μL, if the concentration is too high, it should be diluted appropriately
②: Insert fragment length should not be too short, try to be longer than 200bp, fragments smaller than 200bp are recommended to be assembled by fusion PCR and other techniques first
③: The reaction is recommended to be carried out in a PCR machine or other equipment with precise temperature control.
④: The reaction system is recommended to be configured on ice.
7. After recombination, it can be directly used to transform competent cells. Take 5-10μL of the solution and add it to 100μL of competent cells to transform. Alternatively, the reaction solution can be stored at -20°C, and it is reported that it can be stored for 1 week.
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The chemical transformation scheme of Bacillus subtilis we use
1. Weigh 70 g K2HPO4, 30 g KH2PO4, 10 g (NH4)2SO4, 5 g sodium citrate dihydrate, 1 g MgSO4·7H2O, dissolve them in distilled water, add water to 500 mL, which is 10x minimum salt solution.
2. Weigh 20 g of glucose and dissolve it in 100 mL of water to prepare a 20% glucose solution.
3. Dissolve 0.5 g of hydrolyzed casein in 10 mL of water to prepare 5% hydrolyzed casein solution.
4. Weigh 1 g of yeast extract and dissolve it in 10 mL of water to prepare 10% yeast juice.
5. Autoclave the prepared solution at 115°C for 20 minutes.
6. Prepare 20ml 1mol/L CaCl2 solution.
7. Prepare 20ml 1mol/L MgCl2 solution.
8. Prepare several Erlenmeyer flasks (recommended 250mL), and autoclave at 121°C for 20 minutes together with the above MgCl2 solution and CaCl2 solution.
9. Prepare 10ml 10mg/ml tryptophan solution, use filter to sterilize, and store in a dark place.
10. Take a sterilized Erlenmeyer flask and add 10 mL 10×minimum salt solution, 2.5 mL 20% glucose solution, 0.4 mL 5% hydrolyzed casein solution, 1 mL 10% yeast juice, 0.5 mL 10 mg/mL tryptophan solution, add 85.1 mL of water to make up to 100 mL, which is GMI medium.
11. Take a sterilized Erlenmeyer flask and add 10 mL 10×minimum salt solution, 2.5 mL 20% glucose solution, 0.08 mL 5% hydrolyzed casein solution, 0.04 mL 10% yeast juice, 87 mL ddH2O, 0.1 mL 10 mg/mL tryptophan solution, 0.25 mL 1 M MgCl2 solution, 0.05 mL 1 M CaCl2 solution, which is GMII medium.
12. Streak the WB600 bacterial solution on the LB plate one night in advance to obtain a single colony.
13. Pick a single colony on the plate and inoculate it in 5 mL of GM I liquid medium, and culture it on a shaker at 125 rpm at 30°C for 12-16 h.
14. According to the 10% inoculum volume, transfer 2 mL of the bacterial solution to 18 mL of GM I medium, and incubate for 3.5 h at 37°C and 250 rpm on a shaker.
15. Transfer 10 mL of the medium with bacteria from the previous step to 90 mL of GM II medium, and incubate for 90 min at 37°C on a 125 rpm shaker.
16. Add the same amount of 30% glycerin, mix well, and distribute 0.5 mL/tube, and store in the refrigerator at -80℃.
17. When transformation is needed, take it out of the refrigerator and thaw it in warm water (for example, keep it at 45°C for 2 minutes). Note: Only thawing is enough, no need to heat up the bacteria liquid.
18. Add 10 μL of plasmid. (It is recommended that the concentration of plasmid is not less than 80ng/μL)Incubate at a shaker at 37°C at 125 rpm for 90 min.
19. After centrifugation at 10000xg for 1 min, the supernatant was removed, and the bacteria were resuspended and spread on the corresponding resistant LB plate medium.
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Colony PCR for Bacillus subtilis
Since Bacillus subtilis is a gram-positive bacteria with a thick cell wall, we recommend that the picked colonies be cultured in a small amount of LB medium (100μL) with corresponding resistance for more than 30 minutes, and it is recommended not to be less than 1 hour. Then, the bacteria liquid is heated in boiling water for more than 10 minutes, and after the bacteria liquid cools down naturally, PCR identification is performed.
PCR system：Component Volume 2x EasyTaq PCR polymerase supermix 5μL Primer-F 0.3μL Primer-R 0.3μL Bacteria sample 0.3μL ddH2O 4.1μL -
Agarose gel electrophoresis
The preparation process of agarose gel involves the heating and melting of agarose. This process will cause a lot of water to evaporate, so when adding TAE buffer, we usually add 2-3mL more buffer.
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Plasmid amplification
Although DH5α cells are often used for plasmid cloning, we have found in our experiments that the transformation efficiency of DH5α cells for plasmids longer than 10,000 bp is not ideal. In the experiment, we found that BL21 cells can be temporarily used for plasmid amplification for longer plasmids.
CONTACT NPU CHINA
NPU-CHINA is the iGEM team of Northwestern Polytechnical University
Address: 127 West Youyi Road Beilin District, Xi'an,
Shaanxi 710072, China
Contact: npuigem@outlook.com
bilibili: NPU-iGEM
