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PROTOCOL

Ⅰ. Molecular Cloning:

1. Antibiotics

2. Transformation

( 1 ) Add 10μL purified plasmid or ligation system into 50 μL fresh competent cells, which is contained in 1.5 mL centrifuge tube. Then mix gently.

( 2 ) Leave on ice for 30 minutes.

( 3 ) Heat shock at 42°C for 60 seconds.

( 4 ) Leave on ice for 2 minutes immediately.

( 5 ) Add 450 μL fresh LB medium into the centrifuge tube.

( 6 ) Incubate for 1 hours under the condition of 37°C, 200 rpm.

( 7 ) Spread 100 μl~150 culture medium on a LB solid medium plate, which contains appropriate antibiotics.

( 8 ) ncubate overnight at 37°C (about 12~14hours).

3. Plasmid Extraction (Omega Plasmid minipre Kit I)

( 1 ) Pellet 1.5mL bacteria in two clean 1.5 ml microcentrifuge tubes by centrifugation at 10 ,000 x g for 1 min at room temperature. Decant or aspirate medium anddiscard.

( 2 ) Resuspend the bacterial pellet by adding 250µL of Solution I/RNaseA and vortexing(or pipet ting up and down). Complete resuspension (novisible cellclumps) of cell pellet is vital for obtaining good yields.

( 3 ) Add 250µLl of Solution II and gently mixby inverting and rotating the tube several times to obtain a clear lysate. A 2 minutes incubation maybe necessary. Avoid vigorous mixing as this will shear chro mosomal DNA and lower plasmid purity.

( 4 ) Add 350µL of Solution III and mix immediately by inverting the tube several times until a flocculent white precipitate forms.

( 5 ) Centrifuge at ≥13,000 x g for 10 minutes at room temperature.

( 6 ) Add the cleared supernatant by CAREFULLY aspirating it into a clean HiBind®MiniprepColumn (I) assembled in aprovided 2 ml collection tube. Centrifuge for 1 min at 10,000 x g at room temperature to completely pass lysate through the HiBind®Miniprep Column (I).

( 7 ) Discard flow-through liquid and re-use the 2 ml collection tube.Add 500µL of Buffer HB to wash the HiBind®Miniprep Column (I). Centrifuge for 1 min at 10,000x g at room temperatureto completely pass solution through the HiBind®Miniprep Column (I).

( 8 ) Discard flow-through liquid and re-use the 2 ml collection tube. Add 700µL of DNA Wash Buffer diluted with absolute ethanol to wash the HiBind®Miniprep Column(I). Centrifuge for 1 min at 10,000 x g at room temperature to completely pass solution through the HiBind®Miniprep Column (I) .

( 9 ) Centrifuge the empty column for 2 min at 10,000 x g to dry the column matrix.

( 10 ) Place the column into a clean 1.5 ml microcentrifuge tube. Open the lid and put it in the electric dry oven to volatilize alcohol.

( 11 ) Add 60µL 65°C sterile deionized water directly onto the column matrix and let it sit at room temperature for 1-2 m inutes. Centrifuge for 2 min at 13,000 x g to elute DNA.

( 12 ) Mark and store in fridge.

4. Digestion for Confirmation

Reaction condition: one and half hours under 37℃.

Ⅱ. Characterization:

1. Cultivation and induction

( 1 ) Put the tube with competent cell on the ice. Add 10μL plasmid which was sequenced.

( 2 ) Put the tube back to the ice for 30 minutes after mixing.

( 3 ) Put the bacterial cultures in the 42℃ water for 42 seconds. Then put it in the ice for 2 minutes quickly.

( 4 ) 450mL fresh liquid medium were added. Grown at 37℃，200rmp for 1 hour.

( 5 ) 100μL bacterial cultures were maintained on the agar plate with antibiotic at 37℃ for 12 hours.

( 6 ) The single colonies were picked. Add 10mL liquid mediate with antibiotic. Grown at 37℃， 200rmp for 12 hours.Growth of promising bacterial strain: Cell concentration was determined by measuring the optical density.

( 7 ) Add 10μL，30μL，50μL，70μL，100μL 0.1M IPTG separately to the medium. Incubated at 18 ℃, shaken at 200rmp overnight.

2. Preparation of the cell-free extract

( 1 ) The enzyme expression was induced with the most appropriate concentration of isopropylthiogalactoside at 18°C for 3 h. Cells were centrifuged at 8000×g for 5min.

( 2 ) The column was washed two times with 10 ml of PBS buffer.

( 3 ) The cell pellet was suspended with 7.5 ml of PBS buffer and the cells were broken using Homogenizer.

( 4 ) Ultrasonic breaking cell: power 200w, time 5s, interval 2s, frequency 10.

( 5 ) The cell-free extract : The broken cells were centrifuged at 8000×g for 15min. Supernatant is the cell-free extract.

3. Enzyme and protein assays

In the 96 hole plate, each sample was measured in two parallel samples, each with 220μL of the enzyme reaction system, and the 20μL was added to the reaction system. Set the wavelength of 340nm Eliasa, each 10s reading time, reading 15 points, calculate the absorbance rate of change △A with initial velocity method.

When the condition is certain, the amount of enzyme required for catalytic production or consumption of 1 mol NADH per minute is defined as an enzyme activity unit.

Calculation of enzyme activity:

U/mL = △A × K × VT / VS × ε × d

K：Sample dilution multiple
VT: Total volume（ul）
VS: Sample volume（ul）
d = Ratio of color cup light（d=0.5cm）
ε = molar extinction coefficient(ε=6.22 )

The reaction system of leucine dehydrogenase was: 180μL 1mol/L NH4Cl buffer，10μL 0.5M TMA，10μL 4.4 mM NADH，sample 20μL.

The reaction system of formate dehydrogenase was: 180μL 1mol/L PBS buffer，10μL 0.5M Ammonium formate，10μL 4.4 mM NAD+，sample 20μL.

4. Protein SDS-PAGE gel electrophoresis

1. Sampling 160μL in 1.5mL centrifugal tube, adding 40μL 6x protein buffer , boiling water bath 5min, take 20μL as sample.

2. Electrophoresis conditions: 80V, 30min, 120V, 60-80min.

3. Coomassie brilliant blue dye staining at room temperature: 50rmp, 120min.

4. Decolorization: adding Coomassie blue 50rmp, decolorization decolorization liquid, for the night.

5. Catalysis

1. activation radioactivation:500μL fresh liquid bacteria medium were added into 50mL LB medium. Grown at 37℃，200rmp for 12 hours.

2. transfer: 1%-2% was inoculated in 1000mL LB medium (containing 50 μg/mL antibiotic), Grown at 37℃，200rmp for 3h, and the OD was about 0.6.

3. the most suitable concentration of IPTG was added to the culture medium, and induced at 18℃，200rmp for 12h.

4. collect the bacteria, In -80 degree refrigerator preservation.

5. Add the 10mL 1M NH3OH –NH4Cl buffer, smudge cells，add TMA , ammonium formate ，NADH，constant volume to 15mL, Sampling (500mL) with 1, 2, 4, 6, 8, 10, 12, 24, 48,72 ,96h, In -20 degree refrigerator preservation

6. HPLC analysis of L-tert-leucine

1. The melt cells were centrifuged at 13000×g for 10min.

2. take the supernatant of 200μL joined the 1.5ml EP tube, and add 800μL deionized water.

3. The liquid is pumped out through a syringe of 0.22 m, and is added to the glass bottle, which is detected in the liquid phase detector.