1. Plasmid Transformation

Materials:

Competent cells (E. coli DH5α and BL21(DE3)); liquid and solid LB medium; antibiotics.

Method:

Liquid LB Formulation

1. Weigh 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl in a beaker, respectively.
2. Add 800 mL of Deionized Water(DI water) to the beaker and stir with a glass rod to dissolve all the drugs.
3. Adjust the pH to 7.0 with 1 M NaOH.
4. Pour the solution into a measuring cylinder and refill with DI water to 1 L.
5. Sterilize at 121.3 °C, 103.4kPa using autoclave for 20 min.
6. Store at room temperature.

Solid LB Formulation

1. Weigh 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl in a beaker, respectively.
2. Add 800 mL of DI water to the beaker and stir with a glass rod to dissolve all the drugs.
3. Adjust the pH to 7.2 with 1 M NaOH.
4. Pour the solution into a measuring cylinder, add 15 g of agar powder and make up DI water to 1 L.
5. Sterilize at 121.3 °C, 103.4kPa using autoclave for 20 min.
6. When the temperature of the medium is reduced to 50 °C, add the required antibiotics on an ultra-clean table and shake well.
7. Spread the plates.
8. After the medium has solidified (about 10 min), store it at 4 °C.

Plasmid Transformation

1. Label the 1.5 mL Eppendorf tubes.
2. Let competent cells melt naturally in ice-bath for 30 min, and pre-cool the Eppendorf tubes.
3. Blend 50 μL of competent cells in Eppendorf tubes with 1 μL (50 ng) of plasmid gently.
4. Place the mixed plasmid and the competent cells in ice-bath for 30 min.
5. Heat shock for 90 s at 42 ℃ by metal-bath.
6. Transfer the tubes quickly back to ice for 10 min.
7. Incubate the cells in 800 μL of LB medium.
8. Rotate to amplify the cells at 37 °C at 220 rpm for 45 min, followed by centrifuge at 12,000 rpm to precipitate.
9. Discard 800 μL of the supernatant, mix the rest solution, and spread plate on LB (antibiotics-free) solid culture media.
10. Incubate in constant temperature incubators at 37 °C overnight.

2. Colony Amplification

Materials:

Test tubes; liquid LB medium;

Method:

1. Add 3-5 mL of liquid LB medium (with antibiotics) into 10-mL test tubes.
2. Pick 5 different single-colonies with 5 pipette tips, respectively.
3. Drop the tips into 5 different test tubes.
4. Place the tubes on the shaker for incubation.

3. Plasmid extraction and digestion

Materials:

Plasmid extraction kit; restriction endonucleases.

Method:

1. Extraction plasmid according to the plasmid extraction instructions.
2. Add 23 μL plasmid, 3μL 10×H buffer, 2μL XhoI and 2μL NedI to a 1.5 mL Eppendorf tube.
3. Water bath at 37℃ for 1 hour.
4. Metal bath at 75°C for 10sec.

4 .Agarose gel electrophoresis

Materials:

Agarose; TAE; EB.

Method:

1. Add 0.2 g agarose and 25 mL 1×TAE to a conical flask.
2. Microwave to heat the mixture for 1 min to melt.
3. Add 2 μL EB to the conical flask.
4. Insert in the comb, pour the gel, and wait for the gel to be solidified (~20 min).
5. Remove the comb and place the gel in the electrophoresis bath.
6. Add 10 μL marker into the gel and 8 μL sample mixed with 2 μL 5×Loading Buffer.
7. Run the electrophoresis at 100 V for 30 min.

5. SDS-PAGE

1. Make the separating gel: Pipet appropriate amount of separating gel solution (listed above) into the gap between the glass plates. To make the top of the separating gel be horizontal, fill in water into the gap until a overflow. Wait for 30 min to let it gelate. Make the stacking gel: Discard the water and Pipet in stacking gel untill a overflow. Insert the comb without trapping air under the teeth. Wait for 20-30 min to let it gelate.
2. Make sure a complete gelation of the stacking gel and take out the comb. Take the glass plates out of the casting frame and set them in the cell buffer dam. Pour the running buffer (electrophoresis buffer) into the inner chamber and keep pouring after overflow until the buffer surface reaches the required level in the outer chamber.
3. Prepare the samples: Mix your samples(32μL) with sample buffer (8μL Loading Buffer, 5X). Heat them in boiling water for 10 min.
4. Load prepared samples into wells and make sure not to overflow. Then cover the top and connect the anodes.
5. Set an appropriate volt and run the electrophoresis when everything's done.
6. As for the total running time, stop SDS-PAGE running when the downmost sign of the protein marker (if no visible sign, inquire the manufacturer) almost reaches the foot line of the glass plate. Generally, about 2 hours for a 120V voltage and a 12% separating gel.
7. The PAGE glue after electrophoresis was placed in a Petri dish. Add 30mL G250 staining solution, and shake it in a shaker for 30 min, 60rpm at room temperature.
8. Transfer the glue to the decolorizing solution and shake it in a shaker for 1h. Replace the decolorizing solution and continue decoloring until the strip is clear.

6. Resuscitation and Cryopreservation of BL21

1. Removal the frozen strains from the refrigerator at -80 ℃ and defrosted in the ice box.
2. Add 10 μL bacterial liquid and 3 mL liquid LB medium (with antibiotics) into a test tube.
3. Place the tube on the shaker for incubation.
4. Add 800 uL bacterial liquid and 200 μL glycerine into a 1.5 mL EP tube, then place it in the refrigerator at -80 ℃.

7. Protein Expression

1. Add 1ml bacterial liquid to 300mL liquid LB medium(with antibiotics) and put it into a shaker for 10-12 hours(37℃, 180rpm)
2. Add IPTG (the final concentration is 0.1mmol/L) to the anti-LB medium, and then at 15℃, 200 rpm for 12 hours.

8. Protein extraction

1. Prepare the buffer : 200 mL of 20 mmol/L Na2HPO4-NaH2PO4
2. Add all the bacteria liquid into centrifuge bottles and centrifuge them at a flat 8000 rpm for 10 min
3. Pour off the supernatant and wash with 1mL buffer
4. Add 10 mL buffer into each centrifuge bottle and mix well by blowing with a pipette
5. Collect all liquid into a beaker
6. Rinse with 3-5mL of buffer in a flask and pour into a 50-mL beaker.
7. Ultrasonication (5 sec stop 5 sec for 20 min)
8. Centrifuge the liquid at 13,000 rpm for 30 min at 4°C
9. Separate samples of supernatant and precipitate
10. Collect all supernatant and store it at 4°C
11. Resuspend the precipitate in a beaker and store it at 4°C

9. Gas chromatography-mass spectrometry (GC-MS)

Instruments: Agilent 8890-5977B gas chromatography-mass spectrometer
Chromatographic column: HP-5 MS UI 30 m × 250 μm × 0.25 μm
Injector temperature: 280 ℃
Ionization temperature: 230 ℃
Quadrupole temperature: 150 ℃
Column flow: 1.2 mL/min

Temperature program:

Heating rate (℃/min)	Temperature (℃)	Retention time (min)
/	60	1
20	200	2
30	300	5

Transmission line temperature: 280 ℃
Scanning mode: SIM
Quantitative ion: 91
Qualitative ion: 122 65
Standard: 100 μg/mL

10. The detection of enzyme activity

Materials:

KdcA:

10mM Tris-HCl pH=7 140μL
1M MgCl2 10μL
20mM NADH 10μL
100U Adh 10μL
1M Sodium pyruvate 10μL
20mM TPP 10μL
crude enzyme 10μL

Adh1:

10mM Tris-HCl pH=7 160μL
20mM NADH 10μL
10% acetaldehyde 20μL
crude enzyme 10μL

LP (include KdcA and Adh1):

the citric acid / sodium citrate buffer solution pH=6 140μL
1M MgCl2 10μL
20mM NADH 10μL
1M Sodium pyruvate 10μL
20mM TPP 10μL
crude enzyme 20μL

Method:

1. Use a pipette to quickly transfer the solution to the ep tube and mix thoroughly.
2. The reaction was carried out at 20℃ for 5 minutes. The liquid was added to the quartz cuvette.
3. Use the UV spectrophotometer to detect enzyme activity. At the beginning of the measurement, the change in absorbance value at 340nm was recorded.