1. Add 1μL target gene plasmid sbPETase + Pelb into one micro-centrifuge tube. Then add 1 μL target gene plasmid kil into the second micro-centrifuge tube.
2. Add both 2 μL forward primer and 2 μL reverse primer into each micro-centrifuge tube. This step makes sure DNA polymerase could bind to the correct position before extension.
3. Add 5 μL Mix into each micro-centrifuge tube. It provides necessary enzymes for the system.
4. Add 10 μL ddH2O micro-centrifuge tube. It creates a liquid environment for the reaction.
5. Shake three micro-centrifuge tubes and insert them into the PCR Thermal Cycler and set the following steps
6. Denaturation at 98℃ for 15 seconds
7. Annealing at 55℃ for 15 seconds
8. Extension at 72℃ for 1 minute
9. Repeat steps 2-4 for 35 times

1. fold the weighing paper along the diagonal, weigh 0.3g agarose, then pour it into the conical flask.
2. pour 30 ml TAE buffer into the flask and put it in the microwave oven for 3 minutes, until the agarose is completely dissolved.
3. inject 10 μL Ts-GelRed into the liquid.
4. insert the “comb” into the liquid. Let it stand until the gel is solidified.
5. transfer the gel into the electrophoresis chamber, then pull out the “comb” gently. Pour into enough TAE buffer until the gel is submerged.
6. carefully use the pipette to transfer a 3μL marker into the first hole, then add 2μL template DNA in the rest holes.
7. turn on the power and wait for 18 minutes.
8. take out the gel and place it into the UV transilluminator and turn on the UV light.
9. compare with the marker, and determine the length of template DNA.
10. Recover the gel.

1. add 2 μL NcoI and 2 μL XhoI restriction enzyme into one micro-centrifuge tube, then add 2 μL BamHI restriction enzyme and 2 μL XhoI restriction enzyme into another micro-centrifuge tube.
2. add 5μL buffer, 10μL ddH₂O, and 1μL DNA into each micro-centrifuge tube.
3. 37℃ incubation for 1h.
4. Agarose Gel Electrophoresis and gel extraction.

1. add 2 μL Gibson enzyme, 1 μL PCR product, 1 μL plasmid vector, and 6 μL ddH₂O into the micro-centrifuge tube, 37 ℃ water bath for 20 minutes.

1. Transformation
1. Prepare a box of ice and operate all the following steps on the ice
2. Thaw 50μL×4 chemically competent cells DH5α on ice for 1-2 minutes.
3. Mix two tubes of DH5α competent cell(50μL) with precooled recombinant plasmid pET22b-PelB-SbPETase (2μL)
4. Mix the other two tubes of DH5α competent cell(50μL) with precooled recombinant plasmid pRSFDeut-1-kil(2μL)
5. Ice bath for 30 minutes
2. Heat shock
1. Heat shock at 42°C for 1 minute
2. Ice bath immediately for 2 minutes
3. Cultivation
1. Add 900μL LB medium to every bacteria tube
2. Mix the solution uniformly, and incubate it in the shaker at 37℃, 200rpm for 60 minutes
4. Incubation
1. Aspirate the transformed competent cells with one pipette
2. Coat DH5α with pET22b-PelB-SbPETase uniformly on the LB solid media containing Ampicillin
3. Coat DH5α with pRSFDeut-1-kil uniformly on the LB solid media with Kanamycin
4. Turn the dishes upside down and incubate the cells at 37℃ for 12-16 hours.

1. Column equilibrium
1. Insert one spin column into the collection tube.
2. Add 500μL buffer S to the tube.
3. Centrifuge the tube at 12,000rpm for 1 minute.
4. Discard the filtrate in the collection tube.
5. Insert the spin column back into the collection tube
2. Bacteria collection
1. Take 1.5-5mL overnight cultured bacteria into a centrifuge tube.
2. Centrifuge the tube at 12,000rmp for 1 minute and discard the supernatant.
3. Suspension of bacteria
1. Add 250μL buffer SP1
2. Use a vortex machine to entirely suspend bacteria
4. Open lipid bilayers
1. Add 250μL buffer SP2
2. Immediately turn it upside down 5-10 times gently to make the solution is uniformly mixed.
3. Keep it at room temperature for 2-4 minutes
5. Precipitation
1. Add 350μL buffer SP3
2. Immediately turn it upside down 5-10 times gently to make the solution is uniformly mixed.
3. Centrifuge the tube at 12,000rpm for 10min.
4. Collect the supernatant and transfer it to the spin column.
5. Centrifuge it at 12,000rpm for 30 seconds.
6. Discard the filtrate in the collection tube.
6. Washing
1. Add 500μL washing buffer to the spin column.
2. Centrifuge it at 12,000rpm for 30 seconds.
3. Discord the fluid in the collection tube.
4. Insert the spin column back into the collection tube.
5. Repeat steps 1-4 again
7. Elution
1. Centrifuge the empty spin tube again
2. Put the spin column into a clean micro-centrifuge tube.
3. Add 50-100μL elution buffer to the center of the spin membrane.
4. Place the tube at room temperature for 1 minute.
5. Centrifuge it at 12,000rpm for 1 minute.
6. Store the plasmid solution in a tube

1. PCR
1. Add recombinant plasmid(1μL), site-specific mutagenesis primers(2μL×2), enzyme(5μL), and ddH2O(10μL) to a PCR tube.
2. Denaturation at 98℃ for 15 seconds
3. Annealing at 55℃ for 15 seconds
4. Extension at 72℃ for 1 minute
5. Repeat steps 2-4 for 35 times
2. Enzyme Digestion
1. Add DpnI buffer(5μL), DpnI(1μL) and ddH2O(13μL) to PCR tube
2. Water bath at 37℃ for 2 hours
3. Water bath at 65℃ to stop the reaction
3. Transformation
Same procedure as the transformation of vector and target gene to DH5α competent cell
4. Plasmid extraction
5. Transformation
1. Prepare a box of ice and operate all the following steps on the ice
2. Thaw 50μL×4 chemically competent cells BL21(DE3) on ice for 1-2 minutes.
3. Mix two tubes of BL21(DE3) competent cell with recombinant plasmid pET22b-PelB-SbPETase (2μL)
4. Mix the other two tubes of BL21(DE3) competent cell with recombinant plasmid pRSFDeut-1-kil(2μL)
5. Ice bath for 30 minutes
6. Heat shock
1. Heat shock at 42°C for 1 minute
2. Ice bath immediately for 2 minutes

1. Cultivation
1. Pick BL21(DE3) transformants and inoculate them in 20mL LB liquid medium containing Amp and Kana, incubated at 37℃, 200rpm overnight
2. Transfer 1% cultured medium to 2L LB (Amp and Kana)
2. Induction
1. When the OD600 of the cultured medium reaches 0.6, add 0.1mM IPTG to LB liquid medium
2. The culture is incubated at 18℃ and 200rpm for 16-18 hours
3. Buffer preparation
1. 50mL Buffer A: 50 mM Tris-HCI，300 mM NaCl，10 mM imidazole，pH7.6
2. 50mL Buffer B: 50 mM Tris-HCI，300 mM NaCl，250 mM imidazole，pH7.6
3. 50mL Buffer C: 50 mM Tris-HCI，300 mM NaCl，10% glycerin，pH7.6
4. Protein purification
1. Use buffer A to balance the Ni-NTA column
2. Centrifuge the bacteria culture medium at 4500rpm, 4℃ for 15 minutes
3. Add supernatant to balanced Ni-NTA column
4. Rinse unbound protein with buffer A
5. Elute target protein with buffer B
6. Store the target protein in Elution Buffer C
7. SDS-PAGE