Molecular Cloning
【1】PCR Primers were designed by snapgene software and synthesized at BGI Company. PCR was performed by following the manuals of PrimeSTAR Max Premix (2X) (Taraka, R045) or 2 X EasyTaq PCR SuperMix (+ dye) (Transgene, M256). The former was used for construction and the latter was used for identification. 【2】Gel extraction DNA fragment were separated by agarose gel electrophoresis and then recycled by TIANgeo Midi Purification Kit (TIANGEN, DP209-02) or AxyPrep DNA gel Extraction Kit (AXYGEN, AP-GX-250). 【3】Gibson Gibson was performed by following the manual of pEASY- Basic Seamless Cloning and Assembly Kit (Transgene, CU201). 【4】Endonuclease digestion and ligation The endonucleases and T4 ligase were bought from NEB Company and the experiments were performed by following instructions. 【5】Transformation Transformation was performed by following the manuals of Trans5α Chemically Competent Cell (Transgene, CD201) or Trans1-T1 Phage Resistant Chemically Competent Cell (Transgene, CD501). 【6】Plasmid extraction Bacteria were cultured in lysogeny broth (LB) at 37℃ overnight and plasmid extraction was performed by following the manuals of TIANprep Mini Plasmid Kit (TIANGEN, DP103-02) or AxyPrep Plasmid Miniprep Kit (AXYGEN, AP-MN-P-250). 【7】Sequencing Sanger sequencing was done by BGI Company and snapgene software is used to align the sequences.
Expression and Measurements
1. Transform the plasmids into E. coli BL21(DE3) 2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 4ml LB medium with kanamycin. Add 1mM IPTG to all experimental groups as needed. Incubate at 37 degree Celsius in a shaker overnight. 3. Fluorescence. Acquire 1-3 ml bacteria culture after overnight inoculation. Took fluorescence picture under UV illumination. 4. SDS-PAGE. 4.1 Take 1ml of bacterial solution, centrifuge at 12000g for 1min at 4℃, discard the supernatant, add 100ul of pre-cooled RIPA lysate (high), vortex to mix, settle for 5min, centrifuge at 4℃, 12000g for 10min, and take the supernatant which contains the protein extract. After protein quantification by BCA method using commercial test box, adjust the concentration of the protein solution to 1mg/ml. Take 4 parts of the protein solution and 1 part of 5X protein loading buffer and mix it in a boiling water bath for 5 minutes, centrifuge at 12000g at 4°C for 10 minutes, take the supernatant, and store on ice. Use precast gel purchased from commercial company (Transgen, Precast Tris-Glycine Gel) for protein electrophoresis. 4.2 Add marker at 5μl, and add sample on all other lanes with 10ul sample on each lane. Use 100V for electrophoresis. After the electrophoresis, take out the gel, put it in the staining box, add Coomassie Brilliant Blue dye solution to about 3mm below the gel surface, shake on a horizontal shaker, dye for 30min at room temperature, discard the dye solution, wash off the floating color with distilled water, and add decolorization liquid, decolorize on a horizontal shaker until the result is in good condition for taking pictures.