Preparation of basic experimental material

Luria-Bertani medium

Weigh according to the following formula:
- Deposit them all into the flask.
- Add distilled water to expected volume.
- Stir the mix until the solution becomes clear.
-If you need solid medium, add 2.5% agar into the mix. Otherwise, directly proceed to the next step.
- Wrap the sealing film and put it in a high-temperature steam steriliser to sterilise.
- Store at 25℃.(The solid culture medium needs to be divided into the culture plate)

60Luria-Bertani medium

Weigh according to the following formula: - Deposit them all into the flask.
(Sodium acetate solution is specially for TD80's culture media)
- Add distilled water to expected volume.
- Stir the mix until the solution becomes clear.
-If you need solid medium, add 2.5% agar into the mix. Otherwise, directly proceed to the next step.
- Wrap the sealing film and put it in a high-temperature steam steriliser to sterilise.
- Store at 25℃.(The solid culture medium needs to be divided into the culture plate)

20Luria-Bertani medium

Weigh reagents in the following proportions: - Deposit them all into the flask.
- Add distilled water to expected volume.
- Stir the mix until the solution becomes clear.
-If you need solid medium, add 2.5% agar into the mix. Otherwise, directly proceed to the next step.
-Wrap the sealing film and put it in a high-temperature steam steriliser to sterilise.
- Store at 25℃(The solid culture medium needs to be divided into the culture plate)

Preparation of antibiotics

Weigh reagents in the following proportions: -Place all materials in a 15 ml centrifuge tube.
-Add distilled water or anhydrous ethanol to the desired volume.
-Mix the mixture up and down until the powder is completely dissolved.
- Filter the solution into a new sterilized 15 ml centrifuge tube using a 1 ml syringe and an organic filter tip in an ultra-clean bench.
- Store at -20°C.
(Except for chloramphenicol, antibiotics need to be fully defrosted after being removed from the -20°C refrigerator before being added to the culture medium)
- Application:
-For liquid medium: add 1/1000 volume of solution of antibiotic solution to the sterilized medium,mix thoroughly and stored at 4°C.
-For solid medium: after sterilization in an autoclave, wait until the temperature of the container cools down to about 60°C. Then, add 1/1000 volume of antibiotic solution and mix thoroughly.

Preparation of agarose electrophoresis gel

-Add the desired volume of 1x TAE to the conical flask.
-Add precisely weighed agarose at a concentration of 10 g/L.
-Heat the solution in a microwave oven until the solution is clear.
- Remove the conical flask and add 1/10,000 of the total system volume of nucleic acid dye.
- Pour into electrophoresis cartridge, place comb and wait for solidification.
- Carefully remove the comb.

Preparation of brine substrate

- Weigh the reagents in the following proportions: - Add distilled water to 90% of the volume of the reagents weighed.
- Stir the mixture until the powder is completely dissolved.
- Wrap the sealing film and put it in a high-temperature steam steriliser to sterilise.
- Store at 25°C.

Preparation of 50MM culture medium

1. Preparation of component Ⅰ
Weigh the reagents in the following proportions: - Add distilled water to expected volume.
- Stir the mixture until the powder is completely dissolved.
- Put it in the high-temperature steam steriliser to sterilise.
- Store at 25°C.
2. Preparation of component Ⅱ
Weigh the reagents in the following proportions: - Add distilled water to expected volume.
- Stir the mixture until the powder is completely dissolved.
- Put it in the high-temperature steam steriliser to sterilise.
- Store at 25°C.
3. Preparation of component Ⅲ+Ⅳ
Weigh the reagents in the following proportions: - Add distilled water to expected volume.
- Stir the mixture until the powder is completely dissolved.
- Adjust the pH of the solution to 4.5-5.5 with 5 mmol/L sodium hydroxide.
- Put it in the high-temperature steam steriliser to sterilise.
- Store at 25°C.

Molecular Biology Experiment

Preparation of E. coli S17-1 electronporation-competent cells

Material

LB solid medium; LB liquid medium; sterile water; 10% glycerol; conical flasks (500mL); centrifuge tubes (50mL); centrifuge tubes (1.5mL); spectrophotometer; bench-top centrifuge; thermostatic oscillating incubator; ice box.

Protocol

-The wild-type strain of E.coli S17-1 was taken out from the refrigerator at -80 ℃ and activated by streaking in solid LB medium, and then inverted and incubated at 37 ℃ for 12-16 h. The plate was then incubated at 37 ℃ for 12-16 h in a constant temperature incubator;
-Pick a single clone from the activated plate and inoculate it into 5 mL of LB liquid medium, and incubate it overnight at 37℃, 220 rpm on a shaker;
-Inoculate the overnight culture with 1% inoculum into conical flasks containing 300 mL of liquid LB medium, and expand the culture to logarithmic stage (OD600 of 0.4 ~ 0.6) at 37℃, 220 rpm shaker, and then cool the conical flasks on ice for ~ 30 min, during which the flasks were shaken slowly to make the temperature uniform.
- Dispense the cooled logarithmic phase into 6 pre-cooled 50 mL centrifuge tubes and centrifuge at 4,000 rpm for 15 min at 4℃;
-Pour off the supernatant, add 5 mL of pre-cooled sterile water to the centrifuge tubes, gently blow with a pipette to resuspend the cells, then top up with pre-cooled sterile water to 50 mL, cover with a lid and gently turn up and down to make the cells mix well, and centrifuge at 4,000 rpm for 15 min at 4℃;
- Pour off the supernatant, add 5 mL of pre-cooled 10% glycerol to the centrifuge tube, gently blow with a pipette to resuspend the cells, then add pre-cooled 10% glycerol to 50 mL, cover and gently turn up and down to make the cells mixed well, and centrifuge at 4,000 rpm, 4℃ for 15 min;
- Repeat the previous step
- Pour off the supernatant, pipette off the residual supernatant, and resuspend the cells in 6 centrifuge tubes with an appropriate amount of sterile pre-cooled 10% glycerol (about 5 mL) to achieve a final OD600 of 80 ~ 120
-Dispense 50 μL of bacterial solution into pre-cooled 1.5 mL EP tubes, freeze with liquid nitrogen, and then keep them in the refrigerator at -80 ℃.

PCR

General PCR

Goal

Amplification of genes or replacement of promoter strength for sequences

Protocol

- Add in a PCR tube: - Place the tube in the thermocycler and start corresponding program - After the sample temperature decrease to 16℃ , store the result at 4℃/-20℃

Colony PCR

Goal

To determine whether a particular target gene is present in a particular bacterium.

Protocol

- If the length of the target gene fragment does not exceed 5000bp, add in a PCR tube: -If the length of the target gene fragment is more than 5000bp, add to the pcr tube: -If colony PCR is used to validate the use of the Crispr Cas9 knockout gene a large number of colonies need to be detected ,add to the pcr tube: When a large number of colony PCR are required (usually more than four eight-link pcr tubes, i.e., more than 32) we configure more premix in 1.5 mL centrifuge tubes than the number of systems required and then separate them into PCR tubes.
- Place the tube in the thermocycler and start corresponding program After the sample temperature decrease to 16℃ , stop the program and use the results for gene sequencing.

Plasmid extraction

Material

centrifuge tubes (1.5 mL)；TIANprep Mini Plasmid Kit(Buffer BL,Buffer P1,Buffer P2,Buffer P3,Buffer PD,Buffer PW,Buffer EB,Rnase A(10mg/ml),Spin Columns CP3,Collection Tubes 2 ml）；Centrifugal machine；Vortex machine

Protocol

- Add 500μl of equilibrium solution BL to the adsorption column CP3 (adsorption column placed in the collection tube)
-Centrifuge at 12, 000 rpm for 1min, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube.
-Take 1-5 ml of the overnight culture, add it to the centrifuge tube.
-Centrifuge it at 12, 000 rpm for 1min using a conventional tabletop centrifuge, and aspirate the supernatant.
-Add 250 μl of Buffer P1 (with RNase A) to the centrifuge tube with the bacterial precipitate, and resuspend the bacterial precipitate using a pipette or vortex shaker.
-Add 250 μl of Buffer P2 to the centrifuge tube and gently turn the tube up and down 6-8 times to fully lyse the bacteria.
-Add 350 μl of Solution P3 to the centrifuge tube, immediately turn it gently up and down 6-8 times, mix thoroughly.
-Centrifuge at 12,000 rpm for 10 min.
-Transfer the supernatant collected in the previous step to Spin Columns CP3 by pipette.
-Centrifuge at 12, 000 rpm for 60 sec.
-Add 600 μl of Buffer PW (with anhydrous ethanol) to Spin Columns CP3,centrifuge at 12, 000 rpm for 60 sec, and pour off the waste solution in the collection tube.
-Repeat the previous step
-Centrifuge the empty column at 12, 000 rpm (~13, 400xg) for 2 min.
-Open the lid of the column and place it in a ventilated area for 10 min.
-Place the Spin Columns CP3 in a clean centrifuge tube, add 50ul of 70°C dH(2)O.
-Leave it at room temperature for 2min.
-Centrifuge at 12, 000 rpm for 2min. Dischard the column and store the result at -20℃.

DNA extraction from agarose gel

Material

Centrifuge tubes (1.5 mL)；MolPure Gel Extraction Kit(AC Buffer G1,BD Buffer G1, Wash Buffer, Elution Buffer；MolPure DNA Column G1; 2mL Collection Tube G1）；ddH20；Centrifuges; metal baths

Protocol

-Carefully cut the target band on agarose gel and put it in a centrifuge tube.
- Add 300uL of BD Buffer G1 to the tube and heat at 70°C for 10 minutes in a metal bath, mixing with slight inversion every 2-3 minutes.
- Add 100uL of AC Buffer G1 to MolPure DNA Column G1, centrifuge at 13000 rpm for 1 minute and discard the waste solution.
-After the gel dissolve, transfer the liquid to column and put it in the collection tube, 1 minute at room temperature, centrifugate 1 minute at 12000 rpm and discard the waste solution.
- Put MolPure DNA Column G1 back into the collection tube and add 700 uL of Wash Buffer (add anhydrous ethanol) , centrifuge at 12,000rpm for 30s at room temperature and discard the waste solution.
- Add 500 uL of Wash Buffer (add anhydrous ethanol) ,centrifuge at 12,000rpm for 30s at room temperature and discard the waste solution.
- Put MolPure DNA Column G1 back into the collection tube, centrifuge the empty column at 12,000 rpm for 2 min at room temperature, and air-dry it for 5 min at room temperature to remove the Wash Buffer.
- The MolPure DNA Column G1 was placed in a new centrifuge tube and 30 uL of dH(2)O at 70°C was added to the center of the MolPure DNA Column G1 and left at room temperature for two minutes, then centrifuged at 12,000 rpm for 1 minute and the filtrate was collected
- Discard the column and store the result at -20℃.

Gibson assembly

Goal

To assemble the different DNA parts recuperated by PCR

Protocol

- Add reagents as follow into a 200 μl centrifuge tube.Ensure that the system contains a ratio of linear vector concentration to insert fragment concentration between 2:1-3:1 - Keep warm in thermocycler at 50 ℃ for 60 min.
- Store the product at -20 ℃ .

SDS-PAGE

Goal

Separating protein and polypeptide chain according to their molecular weight, and estimating the molecular and concentration of target protein.

Material

Running buffer；SDS-PAGE gel；electrophoresis apparatus；Premixed Protein Marker (low) TaKaRa；5 x protein loading buffer；metal bath

Protocol

-After the initial treatment of the protein sample, 80ul was added to 20ul 5x loading buffer, mixed well, and boiled in a metal bath at 100 °C for 10 minutes.
-Take 10ul sample, 5ul Marker sample protein glue.
-Set 80 V voltage run for 15 min ( at this time the sample is in a straight line ).
-The voltage was changed to 120 V, and the electrophoresis stopped at 1 cm from the bottom of the separation gel.
-After electrophoresis, remove the prefabricated glue soaked in coomassie brilliant blue dye solution, microwave oven low fire dyeing 1min.
-Pour out the staining solution, pour into the decolorization solution to remove the protein glue, microwave heating for 1min.
-Replace the decolorization solution and shake it on the dyeing shaker for 2h.
-Pour off the decolorizing liquid, add ddH2O to no protein glue, stored at room temperature.

Microbial experiment

Electrically transformed Escherichia coli sensory state S17-1

Material

LB medium；LB agar plates with antibiotics (depends)；Crushed ice；Plasmid used to transform；Receptor cell S17-1；Centrifuge tube (1.5 mL); Electrical Conversion Instrument; 37℃ constant temperature shaker

Protocol

-Remove the prepared sensory cells from the -80℃ refrigerator and melt them on ice (about 5 min);
-Add appropriate amount of plasmid or gibson product (2 μL), flick the wall of the tube to mix well, and leave it on ice for 2 min;
-Pipette the mixed sensory cells into a pre-cooled 2 mm shock cup.
-Pipette the mixed cells into a pre-cooled 2 mm electroshock cup and leave it on ice for 5 min;
-Wipe off the water on the outer wall of the shock cup and put it into the slot of the electrotransferometer;
-Apply shock voltage of 2.5 kV for about 5 ms, quickly add 1mL of liquid LB medium into the shock cup, mix well, then aspirate and transfer to a 1.5 mL centrifuge tube.
-Restore the incubation for 1.5 h at 37°C on a 220 rpm shaker;
-Remove the EP tube and centrifuge at 4,000 rpm for 1 min at room temperature.
-Dispose of 850 μL of supernatant by pipette, resuspend the concentrated cells with the remaining supernatant, and spread the cells in LB solid medium (containing antibiotics corresponding to the target plasmid).
-The cells were incubated in LB solid medium (containing antibiotics corresponding to the target plasmid) for 16 h at 37 ℃ in a constant temperature incubator.

Bacterial conjugation

Goal

Transferring a plasmid from a donor bacterium to a recipient bacterium

Material

Donor bacteria (carrying some kind of plasmid) overnight culture medium; recipient bacteria overnight culture medium; 20 LB solid medium;Thermostatic Oscillator

Protocol

-Pipette 20 µL of recipient and donor bacterial fluids and mix them on a 20 LB solid plate.
-Wait for the bacterial solution to dry out
-Place in a thermostatic shaking incubator and incubate overnight at 37 degrees.

Spread plate

Goal

Screening of strains transfected with specific plasmids

Material

smear stick (sterile); 60 LB liquid medium; 60 LB solid medium with some kind of antibiotic (as appropriate); Thermostatic Oscillator

Protocol

-Scratch the bacterial mass with the tip of a pipette gun onto 60 LB solid medium with the antibiotic corresponding to the plasmid.
-Pipette 120ul of 60LB liquid medium onto the medium from the previous step.
-Spread with a sterile applicator until the surface of the solid medium is dry.
-Incubate at 37 °C for 24-48 hours in a thermostatic incubator.

Formate assimilation

Integration of the folD and ftl genes from vibrio natriegens into the genome

Goal

In order to minimize the adverse effects on the growth of H. bluephagenesis TD80 itself while transferring the exogenous gene

Material

E. coli (with pSEVA321-Cas9 plasmid); E. coli (with pSEVA341-G4-Vib-278-ftl-42-folD plasmid); 20 LB solid medium; H. bluephagenesis TD80; 60 LB solid medium (with chloramphenicol and macrolide); 60 LB solid medium; shaker tube (5 ml); applicator stick (sterile); toothpick; colony PCR system; deep well plate; shaker; thermocycler; thermostatic shaker

Protocol

(1) plasmid transfer
Referring to the plasmid transfer procedure in CysNC gene (synthetic Met) knockout, pSEVA341-G4-Vib-278-ftl-42-folD plasmid and pSEVA321-Cas9 plasmid were transferred into H. bluephagenesis TD80.
(2) PCR double validation
-After culturing a single clone with chloramphenicol and spectinomycin on 60LB solid medium, we designed a pair of universal primers at 50-100bp outside the homology arm of the genome and a specific primer inside the genome.
-We first performed colony PCR with the specific primers, and then left the bacteria on 60LB solid medium. (If integration into the genome is successful, the band is about 3600bp; if it fails, there is no band).
-The pcr samples were subjected to agarose gel electrophoresis and the successful strain numbers were recorded.
-Put the sample plate into a constant temperature incubator and incubate at 37℃ for 8-12 hours.
-Verify the successful strains on the sample plate for the second time using universal primers. (If the integration into the genome is successful, the band is about 6800bp; if it fails, the band is about 2500bp).
-The pcr samples were subjected to agarose gel electrophoresis and the number of the successful strain was recorded
(3) De-antibodying
Referring to the de-resistance step in CysNC gene (synthetic Met) knockout, de-resistance experiments were performed on H. bluephagenesis TD80 transfected with pSEVA341-G4-Vib-278-ftl-42-folD plasmid and pSEVA321-Cas9 plasmid (without addition of Met).

Sodium formate assimilation pathway fermentation

Goal

To verify that a plasmid containing an exogenous gene functions in the target strain

Material

H. bluephagenesis TD80 (pSEVA321 containing the target gene) ；H. bluephagenesis TD80；component Ⅰ; component Ⅱ; brine substrate; component Ⅲ+Ⅳ; NaOH (5 mol/L); electric pipette gun, glass pipette (25 ml); conical flask (150 ml); shaking tube (5 ml) ;antibiotics (as appropriate); sodium formate solution（200g/L）; sodium acetate solution(200g/L); IPTG(2g/L,5g/L,10g/L,20g/L,50g/L; as appropriate)

Protocol

-Designed independent experimental and control groups with three parallel sample fermentations for each strain:
The experimental group used H. bluephagenesis TD80 containing the pSEVA321 plasmid with the target gene;
The control group used wild H. bluephagenesis TD80.
-Add the following reagents to the 5 ml shaker tubes in the experimental group: -Add the following reagents to a 5 ml shaker tube for the control: -Use a pipette tip to pick the target monoclonal colonies up and pumped into a shaking tube.
-Place the shaking tube in a thermostatic incubator at 37 °C for 12 hours.
-Add the following reagents into a 150ml shaker in the experimental group: -Add the following reagents to the control 150 ml shaker: -Place the 150ml shaker in a thermostatic incubator at 37 °C for 12 hours.
-For the experimental group add the following reagents into a 150 ml shaker: -For the control group add the following reagents into 150ml shake flask: -Put into 37℃ constant temperature oscillation incubator, incubate for 48 hours.
-Take out the shake flask for post-fermentation treatment.

Dry weight measurement

Material

15ml centrifuge tube, centrifuge, multi-tube vortexer, -80℃ refrigerator, sealing film, lyophilizer, analytical balance

Protocol

-Take 15ml of bacterial liquid from the shaking bottle after fermentation, centrifuge at 9000rpm, 5min.
-Discard the supernatant, add 15ml of water, and resuspend the precipitate in a shaker (2500rpm.10min).
-Centrifuge at 9000rpm for 5min, discard the supernatant.
-Put on the sealing film, tie the holes, and put into -80℃ refrigerator to pre-freeze for 2 days.
-Put it into lyophilizer for 24h
-Weigh.

Sample testing

Gas Chromatography Detection

Material

Esterification tube；analytical balance；chloroform, esterification solution； organic phase filter head； shaker； gas chromatograph； syringe (1 ml)； metal heater

Protocol

-After the fermentation solution was lyophilized, 30-40 mg of the sample was weighed and added into the esterification tube.
-Add 2 mL of esterification solution and 2 mL of chloroform to the esterification tube in a fume hood.
-Heat at 99.9℃ for 4 h, cool.
-Add 1 mL of ultrapure water to the esterification tube and shake in a multi-tube vortexer.
-Let stand for 1h
-Pipette 1 mL of the lower layer of liquid, use the organic phase filter tip to filter and inject into the gas phase bottle.
-Analyze by gas chromatograph.

Liquid Chromatography Detection

Material

organic phase filter head; liquid chromatograph; syringe (1 ml); benchtop centrifuge; centrifuge tubes（1.5ml）

Protocol

-Take 100ul of bacterial solution from the fermented shake flask and dilute it tenfold with 900ul of ddH2O.
-Centrifuge at 12000rpm for 5 minutes.
-Aspirate the supernatant with a 1 ml syringe, filter it with an organic phase filter tip, and inject it into a dry and clean liquid phase vial.
-Prepare a standard with 3 concentration gradients to include the remaining concentration values and filter through an organic phase filter into a liquid phase vial.
- Liquid chromatography was used for analysis.

T-switch

Fluorescence Characterisation

Materials

60LB liquid medium,spectinomycin,chloramphenicol,deepwell plate,strains to be characterized

Protocol

-Add 1ml 60LB, 0.1% chloramphenicol and spectinomycin to the well, pick up the strain and add it to the hole, and then put the deepwell plate in the shaker at the corresponding temperature for 12-14 hours.Usually, for each strain we would prepare two wells in case of emergency.
-Add 1ml 60LB, 10μl bacterial fluid of last step,0.1% chloramphenicol and spectinomycin to the well, then put the deepwell plate in the shaker at the corresponding temperature for 12 hours.
-Take 100μl bacterial fluid of last step, add 400μl deionised water to the bacterial fluid, then centrifuge for 5 minutes, discard the supernatant and add 500μl 1X PBS, take 200μl into the enzyme standard plate to measure fluorescence using microplate reader（absorbance 600, excitation/emission wavelength 488/520）

P34HB

CysNC gene (synthetic Met) knockout

Goal

To replace strain screening pressure and reduce the use of expensive antibiotics in large industrial fermentation systems to reduce costs

Material

E. coli (with pSEVA321-Cas9 plasmid); E. coli (with pSEVA341-gRNA plasmid targeting the CysNC gene); 20 LB solid medium; H. bluephagenesis TD80; 60 LB solid medium (with chloramphenicol and macrolide); 60 LB solid medium; methionine solution ( 20 g/L); shaker tube (5 ml)applicator stick (sterile); toothpick; colony PCR system; deep-well plate; shaker; thermocycler; thermostatic oscillating shaker

Protocol

（1）plasmid transfer
-Remove the bacterial preservation tubes from the -80°C refrigerator and streak E. coli (with pSEVA341-gRNA plasmid targeting the CysNC gene) on LB solid medium containing kanamycin, E. coli (with pSEVA321-Cas9 plasmid) on LB solid medium containing chloramphenicol, and H. bluephagenesis TD80 on 60 LB solid medium.
-Cultivate overnight at 37°C in a thermostatic shaking incubator.
- Add to shaker tubes of expanded culture of E. coli (containing pSEVA321-Cas9 plasmid): Add to shaker tubes of expanded culture of H. bluephagenesis TD80 -Use a pipette tip to pick the target monoclonal colonies up and pumped into a shaking tube.
-Place the shaking tube in a thermostatic incubator at 37 °C for 8-12 hours.
-Remove the shaker tube and pipette 20 µL of E. coli (containing pSEVA321-Cas9 plasmid) bacterial liquid and H. bluephagenesis TD80 bacterial liquid and mix on a 20 LB solid plate.
-Wait for the bacterial solution to dry out.
-Place in a thermostatic shaking incubator and incubate overnight at 37℃.
-Scrape the spliced bacterial mass onto 60 LB solid medium with ampicillin and chloramphenicol with the tip of a pipette gun.
-Pipette 120ul of 60LB liquid medium onto the medium from the previous step.
-Spread with a sterile applicator until the surface of the solid medium is dry.
-Incubate at 37 °C for 24-48 hours in a thermostatic incubator.
-Remove the plate, pick a monoclonal colony with the tip of a pipette gun, and streak it on 60 LB solid medium with ampicillin and chloramphenicol (for secondary screening)
-Place the shaking tube in a thermostatic incubator at 37 °C for 8-12 hours.
-Add to shaker tubes of expanded culture E. coli (containing pSEVA341-gRNA plasmid targeting to CysNC gene): Add to shaker tubes of expanded culture of H. bluephagenesis TD80（containing pSEVA321-Cas9 plasmid）： -Use a pipette tip to pick the target monoclonal colonies up and pumped into a shaking tube.
-Place the shaking tube in a thermostatic incubator at 37 °C for 8-12 hours.
-Remove the shaker tube and pipette 20 µL of E. coli (containing pSEVA341-gRNA plasmid targeting to CysNC gene) bacterial liquid and H. bluephagenesis TD80 (containing pSEVA321-Cas9 plasmid) bacterial liquid and mix on a 20 LB solid plate.
-Wait for the bacterial solution to dry out.
-Place in a thermostatic shaking incubator and incubate overnight at 37℃.
-Scrape the spliced bacterial mass onto 60 LB solid medium with spectinomycin and chloramphenicol with the tip of a pipette gun.
-Pipette 120ul of 60LB liquid medium onto the medium from the previous step.
-Spread with a sterile applicator until the surface of the solid medium is dry.
-Incubate at 37 °C for 24-48 hours in a thermostatic incubator.
(2) PCR double validation
-After growing a single clone on 60LB solid medium with chloramphenicol and macromycin, we designed a pair of universal primers at the periphery of the homologous arm of the CysNC gene at 50~100bp, and a specific primer inside the CysNC gene.
-Colony PCR was first performed with the universal primers, and the bacteria were left on 60LB solid medium(cotaining Met). (If knockdown, the band is around 4200bp; if failure, the band is around 2600bp)
-The pcr samples were subjected to agarose gel electrophoresis and the successful strain number was recorded
-Put the sample plate into a constant temperature incubator and incubate at 37℃ for 8-12h.
-Use the universal primers to verify the successful strains on the sample plate for the second time. (If the knockout is successful, there is no band; if it fails, the band length is around 2500bp)
-The pcr samples were subjected to agarose gel electrophoresis and the successful strain number was recorded
(3) De-antibodying
-Add the following reagents into the deep well plate: (42℃ 1000rpm shaker culture for 12h to pass on the next generation. And because the chloramphenicol resistance carried by pSEVA321-Cas9 is extremely easy to remove, the removal of spectinomycin resistance was verified from the third generation.)
-Pick the bacteria with successful pcr double validation with a toothpick and add them into the wells of deep-well plate
-Laminate the deep-well plate and incubate at 42℃ 1000rpm shaker for 12h.
-Use a pipette to take 5 μL of bacterial solution from each well and add it to 60LB solid medium containing ampicillin (0.1%), spectinomycin (0.1%) and Met (1%).
-Cultivate at 37℃ in a constant temperature incubator for 24 hours.
-Observe the growth of colonies on the medium: if there are colonies growing, it means that the resistance of this generation has not been removed, and repeat the above operation for the next generation; if there are no colonies growing, it means that the resistance has been removed, and proceed to the next operation.
-Use the tip of the pipette gun to isolate the monoclones by streaking the next generation of bacteria on 60LB solid medium containing Met.
-Incubate for 12 hours at 37℃ in a constant temperature incubator.
-Pick out the single clone for the second step of pcr double validation.
-If successful, it means the de-antibody is successful and the knockout is successful, then save the bacteria.
-If unsuccessful, it means that the knockout was successful but the strain reverted to wild type, and you need to re-verify whether the strain on the retention plate reverted to wild type.
-If the strain on the sample plate reverts to wild type, restart from the plasmid transfer step; if it does not revert to wild type, re-perform the de-immunization and repeat the above steps until the knockout is successful and de-immunization is successful.

Tyrian purple

Shake flask fermentation

Materials

Base(with 50g/L NaCl replaced by 68g/L NaBr),Urea solution(200g/L), MgSO4 solution(10g/L),Component II,Component III&IV,Sodium gluconate solution,10g/L Tryptophan,Twain 80

Protocol

-Add 1ml bacterial fluid and IPTG of the design quantity to the flask,then cultivate in the shaker for 48h.
(The temperature needs to be converted during this period)

- Product analysis

(1) 6-Br-Trp
-The LC conditions were as follows: solvent A was Water with 0.1% formic acid; solvent B was Acetonitrile; flow rate 0.4 mL/min; gradient ramp held 5% B for 2 min, ramped to 20% B from 2 to 5 min, ramped to 95% B from 5 to 12 min, held at 95% B until 16 min, then re-equilibrated at 5% B from 16.1 to 20 min. LC/MS data were collected using Agilent MassHunter Workstation LC/MS Data Acquisition for 6500 series Q-TOF (Version 10.1) and analyzed using Agilent MassHunter Workstation Qualitative Analysis (Version 10.0) software.
（2）Tyrian purple
-Two solvent reservoirs, containing (A) water + 0.1% (v/v) TFA and (B) acetonitrile + 0.1% (v/v) TFA, were used to separate the Tyrian purple components, under gradient elution. The gradient elution program is summarized as below. Separation was performed with a flow rate of 0.5 mL min(−1) and the analytes were eluted in approximately 20 min.The final steps, up to 30 min, were included to reach the initial conditions and achieve mobile phase stabilization. Peak purity control and identification of the peaks in standard solutions and samples was performed by means of the diode array detector. The analytes were quantified at 288, 299, 540 and 590 nm.
Gradient elution program (flow rate 0.5 mL/min): reference: [1]Athina Vasileiadou, Ioannis Karapanagiotis, Anastasia Zotou, Determination of Tyrian purple by high performance liquid chromatography with diode array detection,Journal of Chromatography A,Volume 1448,2016,Pages 67-72,https://doi.org/10.1016/j.chroma.2016.04.046.

SOD/PhaP

Protein-induced fermentation

Goal

In order to test whether TD80 imported into the protein pellet could express the protein in the presence of sodium acetate as a carbon source

Material

H. bluephagenesis TD80 (pSEVA321 plasmid containing the target gene); H. bluephagenesis TD80; Component I; Component II; Saline substrate; Component III+IV; NaOH (5 mol/L); electric pipette gun, glass pipette (25 ml); conical flask (150 ml); shaking tube (5 ml); chloramphenicol (25 mg/ml); magnesium sulfate solution (200 g/L); urea solution (200 g/L); cysteine solution (2 mol/L); zinc sulfate solution (2 mol/L); glucose solution (500 g/L); sodium acetate solution (200 g/L); and IPTG (20 g/L and 50 g/L).

Protocol

Designed independent experimental and control groups with three parallel sample fermentations for each strain:
The experimental group used H. bluephagenesis TD80 containing the pSEVA321 plasmid with the target gene;
The control group used wild H. bluephagenesis TD80.
-Add the following reagents to the 5 ml shaker tubes in the experimental group: -Add the following reagents to a 5 ml shaker tube for the control: -Use a pipette tip to pick the target monoclonal colonies up and pumped into a shaking tube.
-Place the shaking tube in a thermostatic incubator at 37 °C for 12 hours.
-Add the following reagents into a 150ml shaker in the experimental group: -Add the following reagents to the control 150 ml shaker: -Place the 150ml shaker in a thermostatic incubator at 37 °C for 12 hours.
-The experimental group was divided into two groups based on the two carbon sources:
The experimental group using sodium acetate as the carbon source added the following reagents to a 150 ml shaker: The experimental group using glucose as the carbon source added the following reagents to a 150 ml shaker: -For the control group add the following reagents into 150ml shake flask: -Put into 37℃ constant temperature oscillation incubator, incubate for 12 hours.
-Take out the shake flask for post-fermentation treatment.

Protein fermentation post-treatment

Dry weight measurement and OD600

Refer to dry weight measurement and OD600 steps in Formate assimilation for protein fermentation post-treatment.

Protein sample processing

Goal

In order to subject protein gel samples to SDS-PAGE

Material

Ultrasonic cell pulverizer, 1×PBS, 100 mMPMSF, tabletop centrifuge, 1.5 ml centrifuge tube, ice

Protocol

-Centrifuge precooled to 4 °C, 12000 rpm, centrifuged for 10 min, discard the supernatant to collect the precipitate
-The bacterial precipitate was resuspended with 1 ml of 1X PBS, and 10 ul of 100 mM PMSF was added;
-The ultrasonic program was set as follows : power 40 %, ultrasonic 5s, interval 8s, total 3min. Ultrasonic crushing
-After sonication, the supernatant and precipitate were collected after centrifugation at 12000 rpm, 4 °C for 10 min, and the precipitate was resuspended with 1X PBS.
-Use immediately or freeze in a -80 °C refrigerator, remove and thaw when needed