Experiments

Extraction of Plasmids

1. Take 1.5-5.0 mL of overnight bacterial culture, add it to a new 2 mL centrifuge tube, and centrifuge at 6000 rpm for 5 minutes at room temperature to pellet the bacteria. Discard the supernatant.
2. Add 250 µL of Buffer K1 to the tube and vigorously vortex the bacterial pellet to completely disperse the cells.
3. Add 250 µL of Buffer K2 to the tube and gently invert the tube 5-10 times to mix. Let it stand at room temperature for 2-5 minutes.
4. Add 350 μL of Buffer K3 to the tube, and gently invert the centrifuge tube 10-20 times until a white flocculent precipitate forms.
5. Centrifuge at 12,000 rpm for 10 minutes at 4°C, or centrifuge at room temperature at 12,000 rpm for 3-4 minutes.
6. Carefully transfer the supernatant from the centrifuged cells into the adsorption column, and centrifuge at 12,000 rpm for 30 seconds at room temperature to allow the lysate to completely pass through the column. Discard the filtrate in the collection tube.
7. Place the column back into a 2mL collection tube, add 700 μL of Buffer KW to the column, centrifuge at 12,000 rpm for 1 minute at room temperature, and discard the flow-through.
8. Insert the column back into a 2 mL collection tube. Centrifuge at 14,000 rpm for 1 minute at room temperature to remove any residual liquid from the column matrix.
9. Place the column into a clean 1.5 mL centrifuge tube, add 50 µL of Buffer KE to the center of the column membrane, leave it at room temperature for 5 minutes, and centrifuge at 13,000 rpm for 1 minute to elute the DNA.
10. Plasmid DNA is stored at 4°C.

Cultivation of Bacteria

1. Transform the plasmids BsAld-CsAlaDc, gnp1, PPK, and gmas into BL21 E.coli competent cells, and the empty plasmid into DH5α E.coli competent cells.
2. Place the bacteria with plasmids BsAld-CsAlaDc, gnp1, PPK, and gmas on solid LBs which contain amplify to culture them, and place the bacteria with the empty plasmid on solid LBs which contain kanamycin to culture them.
3. Pick the best monoclonal colony of each kind of bacteria, and put the picked colony of bacteria with plasmids BsAld-CsAlaDc, gnp1, PPK, and gmas into liquid LB which contain amplify, the picked colony of the bacteria with empty plasmid into liquid LB which contain kanamycin.

PCR

1. Use SnapGene to design primers for the empty plasmid and plasmids BsAld-CsAlaDc, gnp1, PPK, and gmas:

2. Add 8μl ddH2O, 10μl mix, 0.5μl forward primer, 0.5μl reverse primer, and the template of the empty plasmid and plasmids BsAld-CsAlaDc, gnp1, PPK, and gmas into tubes respectively.
3. Put these tubes into PCR machine with the temperature of 58℃. After the process of PCR, check if the results match the expected ones, BsAld-CsAlaDc with 2636 bp, gnp1 with 215 bp, PPK with 2095 bp, and gmas with 1395 bp.

Golden Gate Assembly

1. Add 1μl PGGA, 2μl T4 DNA Ligase Buffer (10X), 2μl NEBridge Golden Gate Enzyme Mix, and 9μl H2O into a tube.
2. Put the tube into a thermal cycler, setting “(37 C, 1 min to 16 C, 1 min) times 30 to 60 C, 5 min.”
3. Transform 2μl into 50μl BL21 E. coli competent cells.
4. Use SnapGene to design primers of this full plasmid:

5. Test if this full plasmid can have the expecting base pair by DNA electrophoresis, with 214 bp.

Fermentation

1. Prepare the fermentation medium with tryptone 10 g/L, yeast extract 5 g/L, MgCl2·6H2O 10 mmol/L, (NaPO3)6 150mmol/L, and glucose 15g/L.
2. Place the bacteria with full plasmid in the fermentation medium.
3. Add different amount of IPTG, 0μl, 0.25μl, 0.5μl, 0.75μl, and 1μl, to each bacteria culture tube.
4. Send these samples to commercial laboratories to have a test of HPLC: Centrifuge the fermented broth at 12,000 rpm, collect the supernatant, pass it through a 0.22 µm filter membrane, and use a ZORBAX Extend 80A C18 4.6x250mm, 5µm column for HPLC with the following program: