Protocol

1.LB Liquid Culture Media

Materials for 100mL:
Tryptone: 10g
Yeast extract: 5g
Sodium chloride (NaCl): 10g
Steps：
1)Measure 10g of tryptone, 5g of yeast extract and 10g of NaCl. Place them in a 100mL sterilized bottle.
2)Place the bottles into the autoclave for sterilization, under 121°C for 20min.
3)When the culture medium is cooled to 50℃-60℃, add 300μL of corresponding antibiotics, shake the container and mix well.
4)Pour the culture solution onto the petri dish and let the culture solution set before use.
5)Keep the bottles under the room temperature. The solution can be preserve under room temperature for a maximum of 7 days.

2.LB Solid Culture Media

Materials for 100mL:
Tryptone: 10g
Yeast extract: 5g
Sodium chloride (NaCl): 10g
Agarose: 15g
Steps:
1)Prepare the LB liquid medium according to the LB liquid medium formula. Before autoclaving, add 300mL LB liquid medium into a 500mL Erlenmeyer flask and add 5g agarose at the same time.
2)After autoclaving, put on gloves, take out the culture medium, and shake the container to mix agarose thoroughly (the temperature of the culture medium is very high at this time, be careful of burns).
3)When the culture medium is cooled to 50℃-60℃, add 300μL of corresponding antibiotics, shake the container and mix well.
4)Pave a plate (5mL medium/60mm petri dish).
5)Shelf life: 30 days

3.Gel electrophoresis

Materials for 50mL gel:
TAE: 50mL
Agarose [15g/L]: 1.5g
Steps:
1)Add 1.5g agarose into 50mL TAE.
2)Heat the solutions until it becomes transparent.
3)Cool the solutions to room temperature and pour them into a mold.
4)Move the gel plate into electrophoresis tank and soak it into buffer solution. Set baffle and comb in the tank, and seal the baffle inside, until the gel is cooled to about 50˚C.
5)Remove the baffle and vertically pull out the comb.
6)Prepared 5μL marker with 1μL Gel Red, and mix dye . Load marker and samples vertically and slowly with pipetting. Immediately start the electrophoresis with a voltage of 120V.

4.DNA PCR

Materials for 50μL PCR:
Taq Enzyme: 25μL
Template: 1μL
Primer 1 : 1μL
Primer 2:1μL
Double Distilled Water: 22μL
Steps:
1)Add 22μL of Taq enzyme, 1μL of templated DNA, 1μL of primer 1, 1μL of primer 2, and 22μL of double distilled water into a PCR tube.
2)The PCR process: preheat at 98℃ for 3min; Replicate the following steps for 30 times: heat at 98℃ for 10s, anneal at 55℃ for 15s, extend at 72℃ for 50s (at a speed of about 5sec/kb); extend at 72℃ for 5min.
3)Store the product at 4℃.

Table1 Primers sequences for DNA PCR

5.Colony PCR

Materials for 50μL:
Prime STAR Max: 25μL
Primer 1: 0.2-0.3μL
Primer 2: 0.2-0.3μL
Template：1μL
Double distilled water: refill the quality up to 50μL
Steps:
1)Prepare a few 0.2mL PCR tubes with 20μL ddH2O in each.
2)Choose several single colonies from the agar gel medium to drop in each tube via pipette tips, and stir the mixture thoroughly.
3)Preparing a 50μL PCR system in PCR tubes: 25μL PCR Mix Master, 1μL suspension, 0.2-0.3μL of each primer mix. Use double distilled water refill the quality up to 50μL.
4)Protocol of Colony PCR: preheat at 98℃ for 3min; Replicate the following steps for 30 times: heat at 98℃ for 10s, anneal at 55℃ for 15s, extend at 72℃ for 5s/kb; extend at 72℃ for 5min; Store the products at 16℃.

Table 2 Primer sequences for colony PCR

6.Plasmid extraction:

Material:
Buffer P1: 250μL
Buffer P2: 250μL
Buffer P3: 350μL
Double distilled water: 30μL
Buffer PW: 500μL
Steps:
1)Take 1-5mL of the bacterial solution cultured overnight (12h-16h), add it to a centrifuge tube, and centrifuge at 10,000 rpm (11,500 ×g) for 1min. Discard the culture medium, invert on absorbent paper to absorb the residual liquid.
2)Add 250μL Buffer P1 (check whether RNase A has been added to Buffer P1 first) into the centrifuge tube with bacterial precipitation and mix with pipette or vortex oscillation.
3)Add 250 μL Buffer P2 and gently invert it for 8-10 times to fully crack the bacteria.
4)Add 350μL Buffer P3 and immediately gently invert the solution 8-10 times to completely neutralize Buffer P2. A white flocculent precipitate should appear. Centrifuge at 12,000 rpm (13,400 ×g) for 10min.
5)Place a FastPure DNA Mini Column which is an adsorption column in a collection tube 2mL. Carefully transfer the supernatant of Step 4 to the adsorption column with a pipette, and centrifuge at 12,000 rpm (13,400 ×g) for 30-60s. Drain the waste liquid from the collection tube and put the adsorption column back into the collection tube.
6)Add 500μL Buffer PW1 to the adsorption column, centrifuge at 12,000 rpm (13,400×g) for 30-60s, discard the liquid waste and put the adsorption column back into the collection tube.
7)Add 600μL Buffer W2 (please check that it has been diluted with anhydrous ethanol) to the adsorption column, centrifuge at 12,000rpm (13,400×g) for 30-60s, discard the waste liquid and put the adsorption column back into the collection tube.
8)Repeat Step 7.
9)Centrifuge at 12,000rpm (13,400×g) for 1min to dry the adsorption column.
10)Place the adsorption column in a new sterilized 1.5mL centrifuge tube, and add 30-100μL double distilled water to the membrane center of the adsorption column. The DNA was eluted at room temperature for 2min and centrifuged at 12,000rpm (13,400×g) for 1min.
11)Discard the adsorption column and store the DNA products at -20℃ to prevent DNA degradation.

7.Preparation of competent cells:

Material:
BT Media: 50ml
LB liquid culture media: 10-15ml
Bacteria solution: 0.5ml
Steps:
1)Mark the bacteria on a LB plate and place the plate in a 37°C incubator for 12-16h until the colony grew to 1-2mm in diameter.
2)Inoculate a normal growing colony into 10-15ml liquid LB medium, and put it on a 37°C shaking table (≥250rpm) for 12-16h.
3)Take 0.5ml of the activated bacterial solution and inoculate it into 50ml of prepared BT Media. Fully oscillate it on a shaking table at 37°C until OD600=0.5-0.6.
4) Transfer the bacterial solution to 50ml polypropylene plastic centrifuge tube and place it on ice for 5-10min.

8.E.coli transformation:

Material:
LB solid culture media
LB liquid culture media: 700μL
Steps:
1)Take competent E. coli tube out of -80°C and thaw on ice.
2)Add DNA sample and mix gently. Leave on ice for 30min.
3)Heat shock the tube by 42°C water bath for 45s, quickly put it back on ice for 2min, add 700μL sterile LB medium without antibiotic and mix well.
4)Grow in 37°C shaking incubator for 1h (160-225rpm), plate some of the transformation on LB agar plate containing appropriate antibiotic, and stand at 37°C until the liquid is absorbed, then invert and culture overnight at 37°C.

9.C. tyrobutyricum transformation:

Material:
Phosphate buffer solution 50 mM：1mL
Phosphate buffer (PBS, pH 7.0): 600mL
Thiamphenicol: 15μL/mL
RCM plate containing 15μL/mL methylphenicol and 250μg/mL D-cycloserine
RCM medium containing 15μg/mL methyl sulfone
Dried RCM agar plate
Steps:
1)Culture E. coli CA434 with target plasmid, donor cells, in LB medium with 30 μL/mL chloramphenicol until the optical density (OD600) is 1.5-2.0.
2)Take 3mL E. coli CA434 cells, centrifuge at 4,000×g for 2min, and wash twice with 1mL 50 mM PBS.
3)Recapitulate the collected donor cells with 0.3mL of chloramphenicol until OD600 reaches 2.0-3.0.
4)Coat the cell mixture on a dry RCM agar plate and incubate in an anaerobic bag at 37℃ for 20h.
5) Take the colonies grown on the plate and dissolve with 600mL PBS. Re-coat the cell solution on an RCM plate containing 15μL/mL Thiamphenicol and 250μg/mL D-cycloserine. Culture anaerobically at 37℃ for 48-96h until obvious colony growth is observed.
6) Randomly select 10 single colonies from the plate and culture in RCM medium containing 15μg/mL Thiamphenicol at 37℃ for 8-10 h.
7) Take 1μL of the culture for colony PCR. After colony PCR confirmation, take appropriate amount of bacterial solution and expand in fresh RCM medium containing 15μg/mL Thiamphenicol.

10.SDS-PAGE

Steps:
1)Take bacteria solution freshly cultured to OD600=2.0, centrifuge at 10000×g for 1min, and remove the supernatant.
2)Rinse with 1mL PBS buffer at pH 7.0, centrifuge at 10000×g for 1min, remove the supernatant, and then re-suspend with 0.5mL PBS buffer.
3)Break the cells with 450W ultrasound for 10min, and centrifuge at 10000×g for 5min to remove the remaining cell debris and insoluble matter.
4) Add 160μL supernatant to 4×SDS protein electrophoresis buffer and heat at 100℃ for 10min. After cooling to room temperature, samples were ready for protein electrophoresis.

11.Assay of cell metabolites by HPLC

Steps:
1)Add 0.5mL bacterial solution sample from 700mL medium to a 2mL centrifuge tube and centrifuge at 10,000rpm for 1min.
2)Filter the supernatant into a membrane with a pore size of 0.22 micron in the chromatographic sample bottle. Measure the concentrations of carbohydrate, butyric acid, butanol and acetic acid by high performance liquid chromatography (HPLC). Use Aminex HPX-87H organic acid analysis column (100 mm×7.8 mm, BioRad, Marnes-la-Coquette) to determine the products and keep the mobile phase of H2SO4 at 5mM at 65℃ with a flow rate of 0.6mL/min.

12. Determination of cell catalytic products by gas chromatograph

Steps:
1)Take the enzyme-catalyzed product dissolved in cetane out of the membrane.
2)Inject 1μL of the product sample into the gas chromatography with the column model RTX-1 (25 m×0.32 mm×0.50 um, Restek).
3)Set the inlet temperature at 250℃, the temperature of the hydrogen ion flame detector at 280℃, and the initial column temperature at 180℃ for 2 min, and then heat up to 200℃ at 5℃/min for 5min. Then heat up to 250℃ at 30℃/min for 2min.