Team:HUST-China/wetlab/protocols
protocols
1. Making competent cells of E.coli WM3064
a. Inoculate E.coli WM3064 into DAP-LB, cultivate the bacteria at 37℃, 200rpm for 6~8 hours
b. Streak the bacteria on DAP-LB plate medium at 37℃.
c. Pick a single colony and cultivate in 10mL DAP-LB overnight.
d. Transfer 10mL bacterium solution into conical flask with 100mL DAP-LB and cultivate the bacteria. When OD600 increases to 0.4~0.6, put the conical flask in the ice.
e. Put CaCl2-glycerol solution, two 50mL sterilized centrifuge tubes and some 1.5mL EP tubes in the ice.
f. Divide the bacterium solution into to 50mL sterilized centrifuge tubes.
g. Centrifuge at 4℃, 4000rpm for 8 minutes. Discard the supernatant.
h. Add 30mL CaCl2 and resuspend.
Repeat g and h for 4 times.
i. Add 2mL CaCl2- glycerol solution and resuspend. Divide the bacterium solution into 1.5mL EP tubes. Store the competence at -80℃.
2. Transformation of wm3064(2,6-diaminopimelic acid deficiency type)
a. Add 50μl competence wm3064 and 1μl plasmid into an EP tube.
b. Put the tube in the ice and incubate the cell for 30 minutes.
c. Take a heat shock(42℃) for 80 seconds.
d. Take an ice-bath for 3~5 minutes.
e. Add 300μl DAP-LB into the EP tube and incubate the cell at 37℃, 200rpm for 40 minutes.
f. Add 200μl solution in a plate with kanamycin and DAP. Cultivate these bacteria overnight.
3. Conjugation
a. Inoculate Shewanella MR-1 and wm3064 (possess the targeted plasmid).
b. Add 500μl Shewanella MR-1 and wm3064 into a 1.5mL EP tube, mixing the bacterium solution.
c. Centrifuge the solution at 5000rpm for 10 minutes. Discard the supernant.
d. Resuspend the bacteria with 1000mL DAP-K+-LB. Take a 30℃ water bath for 1 hour.
e. Add 200μl solution in a plate with kanamycin.
4. Real-Time Quantitative PCR
(1). Normalize the primer concentrations and mix gene-specific forward and reverse primer pair. Each primer (forward or reverse) concentration in the mixture is 5 pmol/μl.
(2). Set up the experiment and the following PCR program on ABI Prism SDS 7000. Do not click on the dissociation protocol if you want to check the PCR result by agarose gel. Save a copy of the setup file and delete all PCR cycles (used for later dissociation curve analysis). Please note the extension steps are slightly different from described in our paper.
a. 50℃ 2 min, 1 cycle
b. 95℃ 10 min, 1 cycle
c. 95℃ 15 s -> 60℃ 30 s -> 72℃ 30 s, 40 cycles
(3). A real-time PCR reaction mixture can be either 50 μl or 25 μl. Prepare the following mixture in each optical tube.
| 50 μl real-time PCR reaction mixture | 25 μl real-time PCR reaction mixture |
|---|---|
| 25 μl SYBR Green Mix (2x) | 12.5 μl SYBR Green Mix (2x) |
| 0.5 μl liver cDNA | 0.2 μl liver cDNA |
| 2 μl primer pair mix (5 pmol/μl each primer) | 1 μl primer pair mix (5 pmol/μl each primer) |
| 22.5 μl H2O | 11.3 μl H2O |
(4). After PCR is finished, remove the tubes from the machine. The PCR specificity is examined by 3% agarose gel using 5 μl from each reaction.
(5). Put the tubes back in SDS 7000 and perform dissociation curve analysis with the saved copy of the setup file.
(6). Analyze the real-time PCR result with the SDS 7000 software. Check to see if there is any bimodal dissociation curve or abnormal amplification plot.
5. Measurement of Electrode-Attached Biomass
(1). The electrode is placed in a 50 mL tube containing 5 mL NaOH(0.2 mol/L).
(2). Vortex for 2 min and incubate in a water bath to lyse cells at 96 °C for 30 min.
(3). The extracts were tested by bicinchoninic acid protein assay kit (Solarbio, China) after being cooled to 25 °C.
6. Electrogenesis
(1). The electrode is placed in a 50 mL tube containing 5 mL NaOH(0.2 mol/L).
(2). Vortex for 2 min and incubate in a water bath to lyse cells at 96 °C for 30 min.
(3). The extracts were tested by bicinchoninic acid protein assay kit (Solarbio, China) after being cooled to 25 °C.
