Team:iBowu-China/Experiments
Experiments
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 GENEWIZ 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 100 ul bacteria culture and centrifuge at 10000g for 1 min. Aspirate the supernatant and resuspend with 100 ul ddH2O to get rid of the fluorescence background from LB medium. Add 100 µl sample solution into a sterile 96-well plate. Measure fluorescence with a microplate reader and then also measure OD600 for normalization.
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 in the first lane on the left 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.
5. Enzyme Activity
5.1 Take 100 ul of bacterial solution, centrifuge at 10000g for 1min at room temperature and discard the supernatant. Resuspend with 100ul ddH2O, and add into this solution 100ul of standard test solution I (containing Phenolphthalein-β-D-glucuronide, and the test box was purchased from Nanjing Jiancheng Biotech company). Incubate the mixture at 37C for 1 hour.
5.2 Observe the solution. pink or purple color indicates positive enzyme activity. Quantitatively the activity can be measured by OD540 reading on a microplate reader, divided by its OD600 reading for normalization of concentration.