Variants were generated through site-directed mutagenesis using PCR method.
1. Standard PCR reaction
Make reaction mix (Total reaction volume: 50 μL)

2. Implement the process of two-step PCR
Mix the reaction mixture by vortexing and briefly centrifuge to collect any drops on the side of the tube.
1). Predenature templates 98 ℃ for 5 min.
2). Amplification cycles are as below:

3). For the first step in two-step PCR, forward primer and downstream primer are added to two PCR tubes respectively. The first step needs to implement 3 cycles.
4). For the second step in two-step PCR, mix PCR reaction solution of the corresponding forward primer and downstream primer and implement for 15 cycles.
5). Both steps need to extend for another 10 min after implementing cycles and store at 4 °C.

3. Validate PCR products through nucleic acid electrophoresis
1). Dissolve 1 g agarose in 100 mL 1XTAE to make agarose gel
2). Mix PCR reaction samples with 10X loading buffer
3). Load samples and marker
4). Run at 120 V for 20 min
5). Image the gel with a gel imager

4. Digest PCR reaction solution with DPNⅠ for 3 h at 37 °C

Transform competent DH5⍺
1. Thaw a tube of DH5⍺ cells on ice for 5 min.
2. Pipette 10 μL digested PCR reaction solution into the tube. Flick the tube carefully for 6 - 8 times.
3. Plug the tube into ice for 30 min.
4. Heat shock for exactly 90 sec at exactly 42 °C.
5. Plug the tube into ice for another 2 min.
6. Pipette 100 μL of the cell mixture onto LB + Amp plate and spread.
7. Cultivate overnight at 37 °C.
8. Select two isolated colonies for each mutation site
9. Make liquid cultures of selected colonies and cultivate for 12 - 14 h at 37 °C in shaking incubator.
10. Send the broth to the sequencing company (Tsingke Biotechnology Co., Ltd. (Nanjing, China)) and use Snapgene to make sequence comparison.

1. Thaw a tube of BL21 cells on ice for 5 min.
2. Pipette 10 μL (50 ng/L) plasmids (extracted from DH5⍺ cells) into the tube. Flick the tube carefully for 6 - 8 times.
3. Plug the tube into ice for 30 min.
4. Heat shock for exactly 90 sec at exactly 42 °C.
5. Plug the tube into ice for another 2 min.
6. Pipette 800 μL liquid LB medium with no antibiotic.
7. Cultivate for 1 h at 37 °C 220 r.p.m. in shaking incubator.
8. Centrifuge the tube for 2 min.
9. Discard 800 μL supernatant and resuspend bacteria with the rest supernatant.
10. Pipette 100 μL of the cell mixture onto LB + Amp plate and spread.
11. Cultivate overnight at 37 °C.

Incubate weed stock
1. Select two isolated colonies for each mutation site from the plate.
2. Make liquid cultures of selected colonies and cultivate for 12 h at 37 °C.
3. Store weed stock at -80 °C.

1. Pipette 2 mL weed stock and add the weed stock into a flask containing 100 mL liquid LB with antibiotic.
2. Add appropriate amount of isopropyl-1-thio-β-D-galactopyranoside (IPTG) to reach final concentrations of 0.1 mM IPTG, when the optical density of cultures at 600 nm (OD₆₀₀) reached 0.4–0.6.
3. Cultivate the bacteria at 20 °C 150 r.p.m. for 24 h.

Protein extraction
1. Prepare enough sterile 50 mL centrifuge tubes to hold bacteria broth.
2. Centrifuge fermented bacteria broth at 8000 r.p.m. for 10 min.
3. Repeat step 1 till flasks are empty.
4. Add 30 mL PBS (50 mM Na₂HPO₄-NaH₂PO₄ buffer, pH 8.0) into each tube.
5. Centrifuge at 8000 r.p.m. for 10 min.
6. Add 20 mL PBS (pH 8.0) into each tube and resuspend bacteria.
7. Sonicate with Intelligent Ultrasonic Processor for 25 min (3 sec on, 5 sec off, AMP 35%) to break cells.
8. Sonicate the cell slurry until it becomes transparent, indicating most cells have lysed.
9. Centrifuge sonicated cell suspension at 8000 r.p.m. for 30 min, at 4 °C.
10. Verify the supernatant through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and store the supernatant at -20 °C.

Use BHET as the only substrate and react at 30 °C and 50 °C.
1. Dissolve appropriate amount of BHET (molecular weight: 254.24) in PBS (pH 8.0) to achieve concentration of 5 mM BHET.
2. Add 2 mL solution prepared in above step into each well of a 24-well well plate.
3. Pipette 100 μL enzyme extracted from each variant (step Ⅴ)
4. Place the well plate on a shaking table at 30 °C and 50 °C for 6 h.
5. Take samples
6. Filter altogether 400 μL reaction solution with organic filter membrane (0.22 μm).
7. Analyze the samples with HPLC.
8. All the experiments are conducted in triplicate.

Use PET film as a substrate and react at 30 °C, 40 °C, 50 °C and 60 °C respectively.
1. Add 2 mL PBS and a PET film (ø = 6 mm) into each well of a 24-well plate.
2. Pipette 100 μL enzyme extracted from selected variants.
3. Add 100 μL enzyme every 24 h, the whole reaction lasting for 5 d.
4. Take samples in the third and fifth day (specific steps in step Ⅵ).
5. Analyze the samples with HPLC.
6. Two variants were selected through statistical normative scores by assigning weights of 10%, 20%, 30%, and 40% to 30 °C, 40 °C, 50 °C, and 60 °C.

All the experimental steps in this session are completely the same as the session above (Ⅶ), but we added BHETase (BsEst) to construct a two-enzyme system to test the degradation effect. The results are presented in the form of TPA concentration and homogeneous TPA is used in PET recycling.

1. Filter the enzyme degradation solution and collect the filtrate for regeneration.
2. Add appropriate amount of 37% HCl to adjust the pH of the solution to 2.
3. Filter the filtrate and wash with deionized water for several times.
4. Dry under vacuum overnight.
5. Dissolve TPA obtained from chemical-enzymatic catalysis in CH₃OH at room temperature.
6. Add 95% H₂SO₄ to the suspension and stir the mixture under reflux for 24 h.
7. Recrystallize the reaction mixture from dimethyl terephthalate (DMT) and wash with CH₃OH.
8. Dry under vacuum to obtain white powder and analyze the product (DMT) with LC-MS.
9. Add DMT (1.39 g) to the vacuum reactor with EG (1.20 mL) and titanium isopropoxide (0.04 mL).
10. Stir the reactor at 160 °C, 200 °C, 210 °C, 260 °C for 1 h, 1 h, 2 h, 2 h, respectively.
11. Dissolve the resulting PET in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, 10 mL) and add dropwise to CH₃OH to remove the catalyst.
12. Centrifuge the solution, dry under vacuum, and collect PET as a white solid.

10 g NaCl + 10 g tryptone + 5 g yeast extract + 1 L deionized water
(Add 2% agar to make LB agar plate)

1. Add 1 g of analytical agarose and 100 mL 1XTAE into the flask which is only used to prepare analytical agarose gel.
2. Microwave until all the agarose is completely dissolved.
3. Cool the flask until its temperature reach 55 °C
4. Pipette 10 μL nucleic acid dye and shake it evenly.
5. Pour mixture into gel mold.