Team:Nanjing NFLS/Notebook
Notebook
1) DNA extraction of microcystis
Use PowerSoil DNA Isolation Kit（#12888-50，MOBIO）
2) Mutation PCR
Thaw Taq, dNTP, primers, template DNA on ice.
To a new PCR tube, add:
Mix solution well.
Place tube in PCR thermocycler. Set thermocycler program:
Inititial denaturation: 5 min at 95℃;
Loop (35 cycles), Denaturation: 30s at 95℃,Annealing: 30s at 60℃,Elongation: 1min at 72℃;
Final elongation: 10min at 72℃;
Store: 10℃.
We use 5μL of the PCR product for electrophoresis and 45μL for purification.
3) Agarose Gel Electrophoresis
Weigh agarose powder and TAE buffer according to a proper portion, and add them to a 100ml conical flask (we usually make 1.5% Agarose Gel).
- Melt the mixture in a microwave until the solution becomes clear (don’t leave the microwave).
- Let the solution cool down to about 40-50℃ and add DNA gel stain (usually we use EB), pour the solution into the gel casting tray with appropriate comb.
- Let the gel cool until it becomes solid.
- PμLl out the comb carefμLly.
- Place the gel in the electrophoresis chamber.
- Add enough TAE Buffer so that there is about 2-3mm of buffer over the gel.
- Pipette DNA samples mixed with appropriate amount of DNA loading buffer (the dye/GeneFinder is in the loading buffer) into wells on the gel.
- Run the gel at 135V for about twenty minutes.
4) PCR products cleanup and fragment recovery from gel using QIAGEN kit according to manual.
- Qiagen MinElute® Reaction Cleanup kit
- Add 5 volumes of Buffer PB to 1 volume of the PCR reaction and mix. It is not necessary to remove mineral oil or kerosene. For example, add 250 μl of Buffer PB to 50 μl PCR reaction (not including oil).
- If pH indicator I has been added to Buffer PB, check that the color of the mixture is yellow. If the color of the mixture is orange or violet, add 10 μl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.
- Place a MinElute column in a provided 2 ml collection tube in a suitable rack.
- To bind DNA, apply the sample to the MinElute column and centrifuge for 1 min. For maximum recovery, transfer all traces of sample to the column.
- Discard flow-through. Place the MinElute column back into the same tube.
- To wash, add 750 μl Buffer PE to the MinElute column and centrifuge for 1 min.
- Discard flow-through and place the MinElute column back in the same tube. Centrifuge the column for an additional 1 min at maximum speed.
- Place the MinElute column in a clean 1.5 ml microcentrifuge tube.
- To elute DNA, add 10 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the membrane, let the column stand for 1 min, and then centrifuge for 1 min.
- If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
- QIAGEN MinElute® Gel Extraction Kit
- Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.
- Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg or approximately 100 μl). For example, add 300 μl of Buffer QG to each 100 mg of gel. For >2% agarose gels, add 6 volumes of Buffer QG. The maximum amount of gel slice per spin column is 400 mg; for gel slices >400 mg use more than one MinElute column.
- Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation. IMPORTANT: Solubilize agarose completely. For >2% gels, increase incubation time.
- After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). Note: If the color of the mixture is orange or violet, add 10 μl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow. The adsorption of DNA to the membrane is efficient only at pH ≤7.5. Buffer QG contains a pH indicator which is yellow at pH ≤7.5 and orange or violet at higher pH, allowing easy determination of the optimal pH for DNA binding.
- Add 1 gel volume of isopropanol to the sample and mix by inverting the tube several times. For example, if the agarose gel slice is 100 mg, add 100 μl isopropanol. Do not centrifuge the sample at this stage.
- Place a MinElute column in a provided 2 ml collection tube in a suitable rack.
- To bind DNA, apply the sample to the MinElute column, and centrifuge for 1 min. For maximum recovery, transfer all traces of sample to the column. The maximum volume of the column reservoir is 800 μl. For sample volumes of more than 800 μl, simply load and spin again.
- Discard the flow-through and place the MinElute column back in the same collection tube.
- Add 500 μl of Buffer QG to the spin column and centrifuge for 1 min.
- Discard the flow-through and place the MinElute column back in the same collection tube.
- To wash, add 750 μl of Buffer PE to the MinElute column and centrifuge for 1 min.
- Discard the flow-through and centrifuge the MinElute column for an additional 1 min at ≥10,000 x g.
- Place the MinElute column into a clean 1.5 ml microcentrifuge tube.
- To elute DNA, add 10 μl of Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the membrane, let the column stand for 1 min, and then centrifuge for 1 min.
- If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
5) Enzyme digestion reaction and reaction of ligation between target gene and plasmid
1. Enzyme digestion
Incubate the tube at 37℃ for about 2hours, then cleanup according to the previous approaches.
2. Ligation
Mixed thoroughly according to above table, and incubate the tube at 16℃ over night. The ligation products were also cleaned according to the previous approaches.
6) Transformation of E.coli cells
1. Preparation of chemically competent E.coli cells
- InocμLate 2ml LB broth with an aliquot (about 50μL)of the desired E.coli from the -80℃ freezer stock of cells.
- Incubate for 2h at 37℃.
- Add the 2ml seed cμLture to 250ml LB broth and grow at 37℃, shaking (about 200rpm) until OD600 of 0.3-0.4 (about 5 hours).
- Pre-cool the 50ml polypropylene tube, 80 EP tubes, CaCl2-glycerine (0.1mol/L CaCl2) and CaCl2- MgCl2 (80mmol/L MgCl2, 20mmol/L CaCl2). Set the centrifuge and prepare the ice tray.
- Transfer the bacteria into the 50ml polypropylene tube. Place it on ice for 10 minutes.
- Centrifuge at 4℃, 4100rpm for 10 minutes.
- Discard supernatant, then place the tube upside down to make sure trace liquid medium runs out.
- Add 30ml of pre-cooled CaCl2- MgCl2 per 50ml of initial liquid medium to resuspend bacteria cell pellet.
- Centrifuge at 4℃, 4100rpm for 10 minutes.
- Discard supernatant then place the tube upside down to make sure trace liquid medium runs out.
- Add 2ml of pre-cooled CaCl2 per 50ml of initial liquid medium to resuspend bacteria cell pellet.
- Transfer to EP tubes (50μL every tube) and store at -80℃.
2. Transformation of E.coli BL21(DE3)
Thaw competent cells rapidly by immersing frozen tubes in a 37℃ water bath after remove from 70℃ refrigerator. Draw about 50μL of the competent cells in a clean tube and add 5μL recombined plasmid, throw on ice for about 30 min. then put them in 42℃ for 90 second, immediately turn on ice for 2min. add 900μL LB medium and incubate in a roller drum at 37℃ fro 1hours. Took 100μL LB medium contain ampicillin, upside down when dried, put in 37℃ incubator over night. Validation by agarose electrophoresis after the plasmid DNA was extracted from transforming Escherichia coli.
7) Determination of the basic resistance of kanamycin
- Kanamycin was prepared in water at 0µg/mL、5µg/mL、10µg/mL、15µg/mL、20µg/mL and stored at -20℃ in 10-µL aliquots. An aliquot was thawed as needed and used once without refreezing.
- Equivalently draw the cyanobacterium at logarithmic growth stage and inoculate in BG-11 liquid medium containing kanamycin with all above concentration respectively, the cyanobacterium solution was cultured under the optimum conditions for one week and observation of growth status was carried out.
- It is determined that the cell of cyanobacterium cannot resist certain concentration of kanamycin if no algae community were found after one week of culture. Basic kanamycin resistance test of Microcystis aeruginosa shows that the algae cannot resist concentration of 5μg/mL and above on BG11. Therefore, we choose 10-15μg/mL kanamycin for screening after shuttle plasmid pPKE2 containing modified GvpA1 gene is transformed into microcysitis aeruginosa.
8) Transformation of microcysis
- Microcysis (FACHB-854) was cμLtured in BG-11 medium until OD730 reached 0.25-0.35 which is measured with a spectrophotometer.
- The cells were prepared for transformation by washing once in 10 mM NaCl and resuspending in BG-11 at 5 x 108 cells per ml (the cells were concentrated 10 times)
- Aliquots of cells (300 or 400 µL) were dispensed to glass test tubes or microcentrifuge tubes, and recombined plasmid DNA in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.6) was added to a final concentration of 2 to 3 µg/ml. Plasmid DNA was stored as a 20 µg/ml solution, so that no more than 50µL was added to cells.
- The transformation mixtures were incubated for various times at 28 to 30°C in a constant-temperature chamber under standard conditions. The key parameter was the time of incubation of the cells with the donor DNA.
9) Microcysis transposon screening
- The cμLture mixture was coating on Millipore membrane (Φ9cm, pore diameter 0.45µm) covered on BG-11 medium and mircrocysis was cμLtured under standard condition with 20 hours.
- Transfer the membrane in BG-11 solid medium which containing 15µg/mL of kanamycin, This plasmid confers kanamycin resistance to transformed recipient cells. Single colony was found after 7-10 days of cμLture, select the growth colony and transfer to BG-11 liquid medium(containing 10-15µg/mL of kanamycin ), shake frequently and cμLture another 7 days.
- 1)The cyanobacteria of best growth were selected and expansively cμLtured step by step (always containing 15µg/mL of kanamycin).
10) Microcysis transposon screening
The selected cyanobacterium was extended cμLture in BG-11 liquid medium. Continuously cμLturing in illuminating incubator with 2000Lux at 25~28℃, Replace cμLture medium with averagely one weeks to maintain the vitality of cyanobacteria, subcμLture for 2-3weeks. It can be used for the following experiment when the OD 730 of cμLture solution reached 0.6.
11) Sample preparation for SDS-PAGE
- 5mL of induced products was centrifuged with 10000rpm for 10 min under 4℃, collecting the cells.
- Resuspend the precipitate with 2mL 0.5M Tris-HCl (pH 6.8), then centrifuged with 10000rpm for 5 min, repeat this step twice.
- Resuspend the precipitate with 5mL buffer for μLtrasonication under 200W, 1second, with 2 second interval, 30 cycles, total times with 6 min. then centrifuged with 12000rpm for 25 min, collected the supernatant and precipitation respectively.
- Resuspend the precipitate with 80µL ddw, add 20µL 5×loading buffer and 5µL DTT, mixed thoroughly. Prepare another tube with the supernatant with the same procedure. Then boiling in 100℃ for 6min for SDS-PAGE detection.
Procedure of PAGE
Preparation of the separation and concentration gels
AP and TEMED shoμLd be the last to added. When it was added, mixed thoroughly immediately, and after the gel polymerization, pμLl out the comb and wash the sample hole with double distilled water.
- Load 5μL samples, Start electrophoresis under 80V until the bromophenol blue moving into separation gel, then increase the voltage to 100V.
- After electrophoresis, strip the gel and immerge in the fixed solution until the bromophenol blue change to faint yellow; replace the fixed solution with staining solution, then dyeing and decolorizing for 4 hours.
- Removing the staining solution, following another decolonization step, it coμLd be watch and took photo after the band of protein was complete clear. The gel coμLd be stored in distilled water.
12) Western Blotting
Apparatus: Apparatus of SDS-PAGE, Electroblotting Apparatus, Power supply, PVDF membrane（Millipore Immobion-P #IPVH 000 10）, Whatman 3MM paper, Additional Tools: Forceps, sponge pad, scissor, gloves, small plastic or glass container, Shallow tray.
Cell extracts were prepared by homogenizing cells or tissues in the lysis buffer (50 mM Tris–HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40) for 45 min. The soluble protein concentration was measured using Bio-Rad Protein Assay (Bio-Rad). The lysates (50 lg) were separated by 12% sodium dodecyl sμLfate (SDS)-polyacrylamide gel (SDS–PAGE) and transferred to PVDF membranes for immunoblotting assay. The membrane was blocked in 5% fat-free dry milk, and probed with antibodies against the interest proteins. The blots were visualized using the enhanced ECL reagent. The levels of protein expression were semi-quantified by optical densitometry using Image J software.
