- Mix 2.5 g of tryptone, 1.25 g of yeast extract, 1.25 g of sodium chloride, and 250 mL deionized water into a 250 mL beaker to make the liquid LB culture medium
- Measure 0.5g of agarose into a separate 200 mL beaker
- Transfer 50 mL of the liquid culture medium from step 1 into a 200 mL beaker to create the 1% LB solid culture medium
- Transfer the 50ml of LB culture medium and the remaining liquid culture medium to suitable containers for high-pressure sterilization
- Sterilize 4 culture medium plates and 4 disposable streaking tools with UV light
- After cooled to 50℃, distribute 50ml of sterilized liquid LB culture medium into 4 sterilized culture medium plates to be solidified into 4 solid LB culture medium plates

- Transfer samples of Top10 competent cell culture from the freezer into an ice box
- Use a streaking tool to dip into the cell culture and inoculate the culture medium with the competent cell culture
- Label the 4 inoculated LB culture medium plates and invert them for incubation
- The next day, use a pipette tip to take 6 colonies of competent cell culture from the solid LB culture medium - Add 1 mL of sterilized liquid culture medium to each of the 6 samples transferred into 1.5 mL centrifuge tubes
- culture at 37℃, 210 rpm for 90 min
- Place into a makeshift icebox for 30 min
- Transfer the six 1 mL samples into 50 mL centrifuge tubes and centrifuge at 4℃，2700 g for 10 min
- Discard supernatant liquid
- Add 300 μL of glycerol and 300 μL of deionized water into each 50 mL centrifuge tube
- Evenly distribute the solution into 100 μL tubes (fill 24 BL21 microfuge tubes and 20 DH5α microfuge tubes)
- Store in the freezer

- Transfer the liquid culture medium into four 50 mL centrifuge tubes each filled with 25mL - Use a pipette to fill each cylindrical centrifuge tube with 10μL of antibiotic solution (1:1000 ratio) and 8.3 μL of pET28 cell culture (1:3000 ratio) - Place 4 cylindrical tubes in a shaker at 150 rpm for overnight incubation at 37℃

- Initial denaturation of the designated DNA strand at 94℃ for 5 min
- Run a thermocycler for 30-35 cycles: - Final extension of the DNA strand at 72℃ for 5 min

- Mix 0.5 g agarose and 50 mL 1 × TAE buffer in a 200 mL beaker
- Melt the solution in a micro-oven for 1 min
- After the solution is cooled to 55℃, add 5 μL nucleic acid dye
- Pour the agarose solution into the casting tray and wait for 15-20 min for the agarose gel to cool completely
- Place the casting tray into the electrophoresis chamber
- Pour TAE buffer until just covering the gel by 2-3 mm
- Add each DNA sample into a designated slot using a micropipette
- Run electricity in the electrophoresis tank for 30 min
- Place under UV light to check the amount and density of each DNA band

- Extract the designated section of DNA from the agarose gel using a small razor blade
- Weigh and place each agarose sample into a 1.5 mL microfuge tube
- Add 1 mL and 600 μL buffer B2 to the two samples respectively
- Place the two samples in the 50℃ water bath for 10 min
- Transfer the contents of the microfuge tubes into separate absorption tubes
- Centrifuge at 12000 rpm for 30 sec
- Discard the liquid
- Transfer the remaining contents of the microfuge tubes into the absorption tubes
- Centrifuge at 12000 rpm for 30 sec
- Discard the liquid and add 500 μL washer solution to the absorption tube
- Centrifuge at 12000 rpm for 30 sec and discard the liquid
- Repeat steps 10-11
- Centrifuge the absorption tube at 12000 rpm for 30 sec
- place the absorption tube in a clean 1.5 mL microfuge tube and add 35 μL Elution buffer to the center of the absorption tube
- Centrifuge at 12000 rpm for 1 min and seal the 1.5 mL microfuge tubes to be placed in the freezer

(pET28 DH5α, W1-pUC57, W1-pET28 a, sp-pUC57, sp-pET28)
- Add 20 mL liquid culture medium to five labeled 50 mL centrifuge tubes
- Add 10 μL of plasmids containing strains culture medium to the designated 50 mL centrifuge tubes
- For the 2 tubes with W1-pUC57 and sp-pUC57 tubes add 20 μL Ampicillin
- For the tubes with pET28 DH5α, sp-pET28, and W1-pET28 a, add 20 μL Kanamycin
- Place the 50 mL centrifuge tubes in a 37℃ shaker for overnight incubation

- Add 1 μL of each enzyme into two 1.5 mL microfuge tubes
- Add 5 μL of buffer to each microfuge tube
- Add 1 μg of extracted DNA to the microfuge tube (one tube with sample 1 pUC19-75, the other with sample 3 pUC19 donor)
- Add 12 μL of deionized water to the microfuge tubes
- Place in 37℃ water bath for 1 hour
- Place in 65℃ water bath for 30 min to stop reaction process

pET28 DH5α, W1-pUC57, W1-pET28 a, sp-pUC57, sp-pET28
- Take the five 50 mL centrifuge tubes out of the shaker and centrifuge at 3000 × g for 15 min
- In separate tubes set up 5 absorption tubes and add 500 μL buffer S
- Centrifuge at 12000 rpm for 1 min
- Discard the supernatant liquid and add 1 mL deionized water to the 5 samples
- Transfer the 5 samples to 1.5 mL centrifuge tubes and centrifuge at 12000 rpm for 2 min and discard the supernatant
- Add 250 μL buffer SP1 to the centrifuge tubes (which contain RNase A) to resuspend the pellets
- Add 250 μL buffer SP2 to the centrifuge tubes and slowly invert and revert the centrifuge tubes 5-10 times to mix the solution
- Add 350 μL buffer SP3 to the centrifuges tube and slowly invert and revert the centrifuge tubes 5-10 times to mix the solution
- Centrifuge at 12000 rpm for 10 min
- Transfer the supernatant liquid to the absorption tubes
- Centrifuge the absorption tubes at 12000 rpm for 30 sec
- Discard the filtrate and add 500 μL of buffer DW1 into the absorption tube
- Centrifuge at 12000 rpm for 30 sec, discard liquid and add 500 μL of washer solution into the absorption tube
- Centrifuge at 12000 rpm for 30 sec and discard the liquid
- Repeat the previous 2 steps
- Centrifuge at 12000 rpm for 60 sec
- Place the column in a clean 1.5 mL centrifuge tube and add 30 μL Elution buffer to the absorption tubes
- Centrifuge at 12000 rpm for 1 min

- Transfer 2 μL of the sample to a new 1.5 mL centrifuge tube
- Add 1 μL pEasy Blunt Clone Vector to each sample
- Add 2 μL of deionized water
- Place in 37℃ water bath for 10 min
- Take out and place into ice bath

- Take one tube of 100 μL competent cell culture BL21 from the freezer and place it in ice box for 5-10 min
- Add 50 μL competent cell culture to each 5 μL plasmid sample
- Place in the ice bath for 30 min
- Place in 42℃ water bath for 90 sec
- Immediately put in the ice bath for 3-4 min to cool down

- The transformation solution was inoculated into 200mL of LB medium containing resistance and cultured overnight on a shaker (37°C, 170rpm);
- 0.1mM IPTG was added for induction when the OD600 is arranging 0.4-0.6, and then the culture was continued at 37°C and 170rpm for 3-4h;
- After centrifuging at 3000g, 120 μL PMS, 3mL cell lysate, and 240mg lysozyme were added, and the bacteria solution which contains lipase was obtained by ultrasonic crushing.

- Using the equation Mass (mg) = Concentration (mM/mL) × Volume (mL) × Molecular Weight to preparing the Buffer A (50 mM Tris- HCI，300 mM NaCl，10 mM imidazole，pH7.6), B (50 mM Tris-HCI，300 mM NaCl，250 mM imidazole，pH7.6), and C (50 mM Tris-HCI，300 mM NaCl，10% glycerin，pH7.6)
- The crude enzyme solution was centrifuged (4500rpm, 15min, 4°C), and the supernatant was added to the Ni-NTA column equilibrated with Buffer A. After washing the unbound protein with Buffer A, the target protein was eluted with Buffer B.
- Proteins were finally stored in Buffer C.

    5× sample buffer :pH=6.8, 0.06mol/L (0.4mol/L) Tris-HCl buffer, containing 25%(50%) glycerol, 2%(10%)SDS, 5%(volume fraction) β-mercaptoethanol and 0.1% bromophenol blue.
    Sample denaturation: The protein solution was mixed with 5× sample buffer at a ratio of 4:1(volume ratio) (if the protein solution concentration is too high, needs pre-desalting treatment), after heating in a 100°C water bath for 3 to 5min, so that the proteins were fully denatured and then centrifuged at 10000 rpm for 5min, and the supernatant was taken for SDS-PAGE.     Separation gel: (1.5M Tris, pH8.8) 90.9g Tris was taken in 400mL deionized water and stirred on a magnetic stirrer until completely dissolved and then the volume was fixed to 500mL. The pH was adjusted to 8.8, filtered, and stored in an ice box at 4°C.
    Concentration gel: (1.5M Tris, pH6.8) take 90.9g Tris in 400mL deionized water, stir on a magnetic stirrer, until completely dissolved, and then the volume is fixed to 500mL. The pH was adjusted to 6.8, filtered, and stored in an ice box at 4°C.     In protein electrophoresis buffer (5×), 15.1g Tris, 94G glycine, and 5G SDS were added to 800mL deionized water and stirred on a magnetic stirrer until completely dissolved, and then fixed the volume to 1L. Stored it at room temperature without pH adjustment and filtration. Taking 100mL, add 400mL of deionized water, dilute to 1×, and use.     Protein sample loading: According to the protein concentration, an appropriate amount of processed protein samples was added into the electrophoretic well cell in turn, and an appropriate amount of protein standard marker was added into another free well cell.
    Start electrophoresis: Switch on the power supply, first adjust the voltage to 75-80V, and then adjust to 120V when the sample reaches the separation gel, maintaining a constant voltage.
    End of electrophoresis and peeling gel: when bromophenol blue migrates to the bottom of the gel, stop electrophoresis; The protein glue was removed and the bromophenol blue strip separating the bottom of the gel was cut off.
    Staining: Protein glue was stained for 1 hour using Coomassie R250 staining solution Decolorization: The stained protein glue was decolorized with decolorization solution until the background was clean and the protein band was clearly visible for photo analysis.
    Decolorization: The stained protein glue was decolorized with decolorization solution until the background was clean and the protein band was clearly visible for photo analysis.

    0.02789g of p-nitrophenol (p-np) was weighed and dissolved in 100mL of solution B (buffer with different pH), and stored in a brown reagent bottle after configuration and stored at 4°C.     0.02, 0.04, 0.06, 0.08, 0.12, 0.16mL of p-nitrophenol mother solution (2mmol/L) was diluted to 4mL with solution B (buffer of different pH), and the absorption value at 410nm was measured successively. The standard curve was drawn with p-C-nitrophenol (0.01, 0.02, 0.03, 0.04, 0.06, 0.08, mmol/L) as the abscissa and absorbance value Y as the ordinate.

    Solution A: 30.0mg of p-nitrobenzoitolate was mixed with 10.00mL isopropanol and stored at 4°C in A brown bottle (a 15mL foil-coated centrifuge tube can be used).
    Solution B: Add 10μl of TritonX-100, then add 100 ml of disodium hydrogen phosphate-hydrochloric acid buffer (pH 3-12) and mix, and store at 4°C.     1mL~1.2mL of solution A and 9mL ~ 10.8mL of solution B(different pH) were mixed in centrifuge tubes, filled and mixed, kept warm in the 37°C water bath for 5min, and evenly divided into 3 to 4 test tubes, each 2.7mL, which is the system solution; 100 μl crude enzyme solution was added into each test tube quantitatively. In the control experiment, the inactivated enzyme solution (boiling at a high temperature for 5min) was added, and the reaction solution was immediately after the enzyme solution was added. The reaction was performed at 37°C for 10 to 15min and was terminated by the addition of 95% ethanol. Absorbance values were measured at 410nm and 310nm, with two to three replicates for each sample and averaged.

    The pH of the buffer solution was the optimum pH measured by it.     1mL~1.2mL of solution A was added to 9mL~10.8mL of solution B (optimal pH) in a centrifuge tube, filled and mixed, kept warm in the 37°C water bath for 5min, and divided into 3 or 4 test tubes, each 2.7mL, namely the system solution. The crude enzyme solution plus 100 μl was added to each test tube, the inactivated enzyme solution (boiling water for 5min) was added to the control group, and the enzyme solution was added as the reaction solution. A gradient was set every 5°C from 25°C to 70°C for 10 to 15min. The reaction was terminated by the addition of 95% ethanol, and the absorbance was measured at 410nm and 310nm, with two to three replicates for each sample, and the average value was taken.