1.Preparing gel for DNA electrophoresis(24 wells 60ml)
Material：
0.6g agarose
60ml TAE(1x)
6µl (10,000x) 4S Green Plus Nucleic Acid Stain
Steps:
1) Add 0.6g agarose into 60ml TAE buffer and mix thoroughly.
2) Heat the container with the solution in a microwave oven.
3) Heat for 30sec and shake it to see if there are precipitates.
4) Repeat step 3 for several times until the solution is transparent.
5) Add 6µl (10,000x) 4S Green Plus Nucleic Acid Stain to the gel solution.
6) Cool the agarose solution to room temperature and pour it into the mould.
7) Settle the baffle, comb in the mould and stand for 12min.
8) After the gel is solidified, remove the comb and the take the gel out of the mould.
2.DNA Electrophoresis
Material:
Gel prepared previously
DNA loading buffer
Samples to be tested
DNA marker
Steps：
1) Add 10µl of DNA loading buffer to each 50µl system in the PCR tube.
2) Mix them thoroughly by shaking the tube.
3) Add 5µl DNA marker into the very first well of the gel.
4) Add 5µl of sample into the consecutive well.
5) Repeat step 4 until all the samples are loaded.
6) Move the gel into the electrophoresis machine and lose the lid.
7) Start the machine with a voltage of 125V and leave it for 25min.
3.PCR
Material:
1µl DNA sample
20µl ddH 2 O
25µl Taq enzyme
2µl forward primer
2µl reverse primer
Steps:
1) Add 25µl Taq enzyme, 2µl forward primer, 2µl reverse primer, 1µl DNA template, and 20µl ddH2O to a new PCR tube, mix them, and place the tube in a 4℃ refrigerator for 10min.
2) Place the PCR tube in a PCR instrument with the following procedures:
| Temperature | Time | Cycles |
|---|---|---|
| 95℃ | 5 minutes | |
| 95℃ | 15 seconds | 35 times |
| 55℃ | 15 seconds | |
| 72℃ | 1 seconds | |
| 72℃ | 3 seconds | |
| 4℃ | 10 seconds |
4. Gibson Assembly (connection, which requires to 4.amplify purified fragments and linearized vectors, usually obtained by PCR)
The reaction is under 50℃ for 15 min
The amount of reaction system is 10µl
Fragments: vector = 3: 1, add ddH2O to 10µl.
Formula:
Fragments: calculated by (0.06×base pairs)/concentration # The concentration was measured after the DNA recovering
Vector: calculated by (0.02×base pairs)/concentration
5.Construct pMTL-Pthl-Dps plasmid
Material:
pMTL-Pthl-Bs2
Pthl-F and Pthl-R
genome of Deinococcus radiodurans R12
Dps-F and Dps-R
Steps:
1) Use plasmid pMTL-Pthl-Bs2 as template, and use Pthl-F and Pthl-R as primers. Then obtain linearized vector（5461bp） pMTL-Pthl by amplifying.
2) Use the genome of Deinococcus radiodurans R12 as template, and use Dps-F and Dps-R as primers. Then obtain fragment Dps（691bp）by amplifying.
3) Use the method of Gibson assembly to connect the fragment with the vector. Transform the constructed plasmid into Clostridium tyrobutyricum. Use colony PCR to verify the plasmid. Primers of colony PCR (806bp): Dps-pF and Dps-pR. The reassembled plasmid obtained at last: pMTL-Pthl-Dps.
6. Construct pMTL-Pthl-aceE plasmid
Material:
pMTL-Pthl-Bs2
Pthl-F and Pthl-R
genome of E. coli DH5α
aceE-F and aceE-R
Steps:
1) Use plasmid pMTL-Pthl-Bs2 as template, and use Pthl-F and Pthl-R as primers. Then obtain linearized vector（5461bp） pMTL-Pthl by amplifying.
2) Use the genome of E. coli DH5α as template, and use aceE-F and aceE-R as primers. Then obtain fragment aceE（2704bp）by amplifying.
3) Use the method of Gibson assembly to connect the fragment with template. Transform the constructed plasmid into Clostridium tyrobutyricum. Use colony PCR to verify the plasmid.Primers of colony PCR (2936bp): aceE-pF、aceE-pR. The reassembled plasmid obtained at last: pMTL-Pthl-aceE
7.Plasmid extraction:
Material:
5 ml Escherichia coli solution CA434
250µl Buffer P1
250µl Buffer P2
350µl Buffer P3
500µl Buffer PW1
600µl Buffer PW2
30 – 100µl elution buffer
Steps:
1) Take 5 ml of bacteria solution, add into a centrifuge tube, centrifuge at 1,000 rpm for 1min, and discard the supernatant.
2) Add 250µl Buffer P1, and mix it with a pipette by oscillation.
3) Add 250µl Buffer P2, and gently turn it upside down 8-10 times.
4) Add 350µl Buffer P3, gently turn it upside down for 8-10 times, and centrifuge at 12,000 rpm for 10min.
5) Put the adsorption column into the collection tube, move the supernatant in 4 to the adsorption column, centrifuge at 12,000 rpm for 30sec, and discard the supernatant.
6) Add 500µl Buffer PW1 to the adsorption column, centrifuge at 12,000 rpm for 30sec, and discard the supernatant.
7) Add 600µl Buffer PW2 to the adsorption column, centrifuge at 12,000 rpm for 30sec, and discard the supernatant.
8) Repeat Step 7.
9) Place the adsorption column back into the collection tube and centrifuge at 12,000 rpm for 1min.
10) Place the adsorption column in a new centrifuge tube, add 30µl elution buffer, set it stand for 2min, and centrifuge at 12,000 rpm for 1min. Throw away the adsorption column use left liquid for further measurement.
11) Measure the concentration of left liquid in centrifuge tube by Nanodrop Nucleic Acid Protein Analyzer: blank control was set with ddH2 O. After cleaning the fiber surface, 2µl of DNA sample was added to the lower fiber surface. Drop the detection arm, the plasmid DNA concentration was measured as 30.3ng/µl. 12) Add 10µl plasmid DNA sample and 20µl ddH2O into a PCR tube with a pipette gun to dilute the DNA sample to 10.1ng/µl for PCR.
8.The transformation of E. coli
Material:
E. coli DH5α
LB medium
Sterile medium
DNA sample
Steps:
1) Remove the E. coli in sensory state from the -80°C refrigerator, and quickly insert it into the ice box to let it thaw.
2) DNA samples were added and gently mixed for 30 min in ice.
3) After heat shocking in water bath at 42°Cfor 90 seconds, quickly put it back in the ice for about 2 minutes, be careful not to shake. 700 μl of sterile medium without antibiotics was added and mixed evenly.
4) Culture it while shaking at 37°C for 1 hour (220rpm), absorb an appropriate volume and evenly coat into the LB agar medium plates containing the corresponding antibiotics. Set upward at 37°C until the liquid was absorbed and then invert for overnight culturing.
9. Cell culture conditions
For E. coli:
Aerobic conditions: 250mL Erlenmeyer flask, liquid volume 15mL, rotate speed 200 rpm, at 37℃, using LB medium
Anaerobic conditions: 15mL centrifuge tube, filled volume 15mL, quiesced (0 rpm), at 37℃,using LB medium
For Clostridium tyrobutyricum:
Aerobic conditions: 100mL anaerobic flask with rubber seal, half filled, rotation speed 100 rpm, at 37 °C, using Reinforced Clostridium Medium(RCM)
Anaerobic conditions: 100mL anaerobic flask with rubber seal, half filled, quiesced in an anaerobic incubator, at 37 °C, using Reinforced Clostridium Medium (RCM)
10.Growth performance assay
The OD600 data were determined by using UV-Vis spectrophotometer, sampling at different times and determining the light absorption values at 600nm.
11.Induction of IPTG protein expression
1) Convert the expression plasmid to E. coli or Clostridium tyrobutyricum. Place the plate on the antibiotic selection plate and incubate overnight at 37°C.
2) Resuspend individual colonies in a liquid culture with antibiotics to produce the starting culture. Inoculate the starting culture into 50mL of expression medium containing antibiotics.
3) Incubate at 37°C with shaking at 220 rpm until OD600 reaches 0.6.
4) For most carrier systems, induce with 250μM IPTG and express proteins at 220 rpm shaking for 24 h at 16 °C.
12.Cell lysis
12.Cell lysis 1) Divide the bacterial solution into 50mL centrifuge tubes. Centrifuge at 6,000 rpm for 10 min at 20°C and pour the supernatant into the waste bin.
2) Resuspend the cell pellet in 10mL 10 mM Tris-HCl (pH 7.5), centrifuge at 6,000 rpm for 10 min at 20 °C and pour out the supernatant. Repeat the steps above.
3) After centrifugation, resuspend the cell pellet in 9.9mL 10 mM Tris-HCl (pH 7.5) and 0.1mL lysozyme and incubate at 30 °C for 20 min.
4) Add 200μl of 100 μM PMSF to the fermentation hroth. Remove 200μl of liquid into a 2mL centrifuge tube and label.
5) Immerse the bacterial suspension in ice and perform sonic treatment with 3 sec sound waves and 3 sec intervals for a total of 30 min.
6) Remove 200μl of whole protein solution from a 50mL centrifuge tube, place it in a 1.5mL centrifuge tube and label it.
7) Centrifuge at 4°C and 6000 rpm for 20 min, remove 200μl of supernatant from a 50mL centrifuge tube into a 2mL centrifuge tube and label. Pour the remaining supernatant into a new 10mL centrifuge tube and wait for ultrafiltration. Resuspend the pellet in a 2mL centrifuge tube with 300μl of pure water and label.
13. Product collection and purification
1) Take an appropriate amount of fermentation hroth, disperse into two 50mL centrifuge tubes, centrifuge at 3500 rpm, equilihrate at 10 °C for 10 min.
2) After centrifugation, pour out the supernatant, take 50μl of the supernatant and label it as sample 1;
Take a certain amount of ultrapure water, resuspend the bacteria, and label it as sample 2;
(1) Add 10mL of ultrapure water to each of the two ultrafiltration tubes, balance, centrifuge at 3500 rpm for 20 min;
(2) Add 10mL of sample 1 to each of the two ultrafiltration tubes, equilihrate, centrifuge at 3500 rpm for 20 min, aspirate the liquid on the filter, and then transfer to a 1.5mL centrifuge tube;
Repeat the operation of step 2 until all samples 1 are processed, the liquid in the 1.5mL centrifuge tube is labeled as sample 3, and the liquid in the 35mL centrifuge tube (i.e., the supernatant after ultrafiltration) is labeled as sample 4;
(3) Add 10mL of ultrapure water to each of the two ultrafiltration tubes, horizontally, at 3500 rpm, centrifuge for 20 min (clean the filter), and immerse the filter in ethanol.
14.Affinity chromatography
1) Take the filler nickel column, and let the tip sit still until the blue resin is completely settled. Then add 10mL of equilihration buffer for equilihration, open the lower plug and drop the liquid.
2) After 10mL of liquid flows out, plug the outlet plug, add the clarified sample after treatment, seal the top cover, slowly and repeatedly flip the column for 1 h (or invert for 1 h, this process is best at 4 °C).
3) Place the tip of the column vertically downwards, allowing the resin to sink to the bottom of the column and attach the EP tube below the tip according to the amount of sample applied.
4) Carefully remove the upper and lower stoppers to collect the dripping liquid. If the load continues after the collection is complete, return to process 2).
5) Wash the column with 10mL of equilihration buffer and then wash the column again with 10mL of wash buffer.
6) Elute the column with 10mL of eluent, collect all the effluent, ultrafiltration concentrate.
15. SDS-PAGE Validation:
Biotec SDS-PAGE Gel Preparation Kit is used. The gel is washed in pure water and then stained with 0.25% Coomassie hright blue to reveal protein bands.
16.Colony PCR
Material:
Target colonies
21µl ddH2 O
25µl 2x Rapid Taq Master Mix
2µl forward primer(10µM)
2µl reverse primer(10µM)
Steps:
1) Add 25µl 2x Rapid Taq Master Mix, 2µl forward primer, 2µl reverse primer, and 21µl ddH2O to PCR tubes each, then use a pipette tip to pick up one target colony and pipette it in those solution each. Mix well, then place the tubes in a 4℃ refrigerator for 10min.
2) Place the PCR tube in a PCR instrument with the following procedures:
| Temperature | Time | Cycles |
|---|---|---|
| 95℃ | 5 min | |
| 95℃ | 15 s | 35 times |
| 55℃ | 15 s | |
| 72℃ | 1 min | |
| 72℃ | 3 min | |
| 4℃ | 10 min |
