3.1 Real-Time Quantitative PCR
1. Normalize the primer concentrations, 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). 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
d. 72℃ 10 min, 1 cycle
3. A real-time PCR reaction mixture can be either 50 μl or 25 μl. Prepare the following mixture in each optical tube.
|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 μ H2O
||11.3 μl H2O
25 μl SYBR Green Mix (2x)
0.5 μl liver cDNA
2 μl primer pair mix (5 pmol/ μl each primer)
22.5 μl H2O
OR 12.5 μl SYBR Green Mix (2x)
0.2 μl liver cDNA
1 μl primer pair mix (5 pmol/μl each primer)
11.3 μl H2O
4. Remove the tubes from the machine, after finishing PCR. 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.
3.2 Detection of Flag
a. Take some Synechocystis in 2ml EP tube and centrifuge at 5800rpm for 15min。Discard the supernatant，add a fresh BG11 medium containing regulatory factors and centrifuge after the 20m, discard supernatant. Wash the cells with 1X PBS;
b. Add 1X SDS Sample buffer (6-well plate per hole 100µl or 500µl) PS: because of the unknown expression, slightly less to increase protein concentration to lysis cells. Immediately scrape the cells off the board and transfer the extract to the micro centrifuge tube.
Put on ice.
c. Ultrasonic crushing 10–15 seconds to complete the cell lysis as well as to cut DNA (to reduce the sample viscosity).
d. Take 20µl samples and heat for 5 minutes under 95–100°c.Then cool the sample with ice.
e. Add 20ul samples to SDS-PAGE (10cm X 10cm)
a. Remove the gel from the glass plate and abandon the lower part of the separation of glue. Make the remaining gel containing the target protein soaked in the membrane buffer to prevent the gel from solidification, and deformate.
b. Wet: Prepare 2 layer of Whatman 3 #filter paper, 1 NC membrane (the size as similar as the gel size) . Put NC membrane in the water for 3 minutes, and then put into the membrane buffer for 10 minutes. (If using PVDF membrane, should soak in 100% methanol previously, otherwise it is difficult to wetting.) (Methanol can be reused.)
c. Put the buffer soaked sponge, 1 layer of whatman3 #filter paper, gel, nitrocellulose membrane, 1 layer of Whatman3# filter paper, sponge in order . Ensure that there is no air bubbles between each layer.
Assembly Order: Turn film clip black (negative)-Sponge-pad-Filter paper-Adhesive-film-filter paper-sponge pad-red face (positive) Remember: Get rid if the bubble between each layer with glass rod.
d. Transfer film: Transfer the clips into the transfer groove and add 4 ℃ pre-cooled membrane buffer. Make the gel surface connected with the negative electrode when nitrocellulose membrane and the positive electrode connect. Transfer film current 350mA, transfer film 45MIN,PS: adhesive in the negative, the membrane close to the positive, the membrane buffer must be pre-cooled. Preferably a day ahead of preparing the buffer and put in the 4 ℃ refrigerator. Filter paper should not be larger than the film ,to prevent short circuit.
e. After the film and gel are clamped and ensure that no bubbles exist between the gel/film and filter .Otherwise it will cause the film to be incomplete. Make sure to wear gloves or touch the membrane with plastic tweezers, because the protein and grease on the hand will affect the film efficiency and make the membrane dirty. In the process of film transfer, especially the high-current rapid transfer, there will usually be a lot of heat. Preferably put the transfer film slot in the ice bath for the trarsmembra.
a. Wash the nitrocellulose membrane with ML TBS for 5 minutes, after the transfer, at room temperature.
b.Place the membrane in an ml buffer and incubate at room temperature for 1 hours.
c. Wash three times with 15ml TBST, 5 minutes each time.
a. Place the membrane and the first antibody(1:1000 buffer diluted)in a 10 ml 5%w/vBSA,1xTBS,0.1%Tween20 and incubate overnight at 4°c without gently shaking.
b. Wash three times with 15ml TBST, 5 minutes each time.
c. Put the membrane with Anti-mouse IgG, hrp-linked Antibody (#7076, by 1:2000 ratio) and Anti-biotin, hrp-linked Antibody (#7075, press 1:1000–1:3000). Incubate at room temperature for 1 hours with a gentle shaking.
d. Wash three times with 15ml TBST, 5 minutes each time.
a. Conjugate of antibody and clean membrane with HRB in TBST for 5 minutes, three times.
b. Dilute part of 2X reagent A and part of 2X reagent B to prepare 1X SignalFire™ ECL Reagent (#6883).Mix evenly.
c. Incubate the substrate with the membrane for 1 minutes and pour out the excess solution (make the membrane remain moist), then wrap it in plastic and expose it under X-ray film.
3.3 Detection of 6×His
1. Weigh and add the bacteria solution, then centrifuge at 5000rpm for 10min.
2. Add lysis buffer for 10-20ml/g bacteria and shake.
3. Ultrasonic crushing, ultrasonic 2s and gap 3s by turns for 5min.
4. Centrifugate at 12500rpm for 30-40min at 4 °C.
5. Take the column firstly loaded with NiSO4, then wash it with deionized water until the effluent is colorless. The leaching-out nickel can be recycled.
6. Add lysis buffer to the column.
7. Add the centrifugation supernatant.
8. Add wash buffer.
9. Add 4ml Elution buffer and gather the liquid with the collection tube (a few drops can be abandoned at first and at last)
10. Add NiSO4, cover for a short-term preservation of the middle of liquid. If it should be long-term stored, NiSO4 needs to be washed off and then add 10% alcohol.
3.4 Lactate detection assay
1. Add 2mL bacteria into 2mL EP tube.
2. Centrifuge at 12000rpm for 1 minutes.
3. Prepare the following mixture in 10mL tubes.
4. Take 0.02mL supernatant into each experimental group.
5. After mixing, incubate at 37℃ for 10 min.
6. Add 2mL into 10 mL EP tube.
7. Detect absorbance (OD530)
|Solution I (mL)
|Enzyme solution (mL)
|chromogenic agent (mL)