AHUT-ZJU-China

Protocol

READING TIME: 63mins

Plasmid transformation TB1

Take TB1 competence and add 1ul plasmid, flick and mix well and place on ice for 30mins

Place at 42℃ for 90s

Place on ice for 3 minutes

Add 200ul LB medium

Incubate on a shaker at 37°C 220rpm for 45mins

Spread the bacterial liquid evenly on the KANA resistant plate and incubate at 37°C for 12-16hr

Induced expression

Pick a single colony from the plate and culture it in 10mL liquid LB medium containing KANA resistance at 37°C and 220rpm in a shaker for 12-16hr

Take 2mL of bacterial liquid and add it to 200mL of KANA resistant medium, and expand the culture for 4hr

Add 1000ul 100mM IPTG 20°C 220rpm and incubate for 8hr

Centrifuge at 10000rpm 4°C for 10mins to collect the bacteria

Add the lysate to ultrasonic, centrifuge to get the supernatant

Turn on the peristaltic pump and wash the nickel column with distilled water

Wash again with PBS

5x imidazole balanced nickel column

Slowly pump the sample into the nickel column

Wash with 50x imidazole to remove impurities

250x imidazole elution target protein

Add the target protein to the concentration tube and centrifuge to concentrate to less than 2mL

Add 2mL desalting solution and centrifuge to less than 1000mL

Prepare 15% SDS-PAGE gel with Coomassie brilliant blue staining

Measuring esterase enzyme activity

1) Prepare 0.1 M phosphate buffer solution, first prepare solution A (0.1M sodium dihydrogen phosphate aqueous solution), weigh 11.998 g of NaH2 PO4 in a beaker, add dissolution and transfer to a 1000 ml volumetric flask, and make the volume constant. B solution (0.1 M disodium hydrogen phosphate aqueous solution), Na2HPO4 14.196 g is placed in a beaker, added to dissolve, and transferred to a 1000 ml volumetric flask, constant volume. Take 17 mL of A solution and 33 mL of B solution, and mix well to obtain a 0.1 M phosphate buffer solution with pH 7;

2) Prepare 50 mL with a concentration of 3 mM p-nitrophenyl acetate (p-NPA), weigh 27.2 mg p-nitrophenyl acetate into a 50 mL burner, add 1.5 mL acetone, stir to dissolve, and transfer to 50 mL Volumetric flask, add water to make the volume;

3) Prepare 100 mL of 1 mM p-nitrophenol (p-NP), weigh 0.139 g of p-nitrophenol into a 100 mL beaker, add 3 mL of acetone, stir to dissolve, transfer to a 50 mL volumetric flask, add water Volume;

4) Prepare different concentrations of p-nitrophenol, as shown in Table 1.1:

Table 1.1 Different concentrations of p-nitrophenol (p-NP) preparation

Serial number	1	2	3	4	5
Concentration	0.05 mM	0.1 mM	0.15 mM	0.2 mM	0.25 mM
p-NP（1 mM）	50μL	100μL	150μL	200μL	250μL
Water	950μL	900μL	850μL	800μL	750μL

5) Measure the absorbance value of each concentration with a microplate reader at a wavelength of 400 nm, and draw a standard curve;

6) Prepare different concentrations of p-nitrophenyl acetate; see Table 1.2

Table 1.2 Preparation of p-nitrophenyl acetate (p-NPA) in different concentrations

Serial number	1	2	3	4	5
Concentration	0.5 mM	1 mM	1.5 mM	2 mM	2.5 mM
p-NPA（3mM）	1mL	2mL	3mL	4mL	5mL
Water	5mL	4mL	3mL	2mL	1mL

7) Add 50μL of phosphate buffer, 100μL of p-NPA of different concentrations, and 50μL of enzyme solution (1μg/mL) to the ELISA plate, and measure the absorbance at 384nm for 6mins.

8) Compare the standard curve and calculate the conversion rate. Enzyme activity is defined as: at room temperature, the amount of enzyme required to hydrolyze 1 μmol of p-NPA acetate per minute is one unit of enzyme activity.

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