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@@ -85,6 +91,7 @@
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+ + FIGURE +
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+
Figure S1. The map of plasmids used for directed evolution + of recombinant plasmids. (A) + pHCM12M-SPC-PETase . This plasmid + contains the gene of IsPETase. (B) pHCM12M-SPC-BsLipA. The + plasmid contains the gene of BsLipA. (C) pHCM12M-SPC-1028. The plasmid contains the gene of Lipase1028.
++
+
Figure S2. The map of pBBR1MCS-tph plasmids for KT2440 strain modification. This plasmid contains the tph clusters including genes encoding the + transcriptional regulator (TphR), TPA transporter + (TpaK), TPA 1, 2-dioxygenase + (TphA), and 1, 2-dihydroxy-3, 5-cyclohexadiene-1, 4-dicarboxylate + dehydrogenase (TphB) from a TPA + degrading P. + Stutzeri. +
++
+
Figure S3. The DNA bands of IsPETase and BsLipA performing agarose gel (1 %) electrophoresis (120V, 30min). The correct band length for IsPETase is 789 + bp and for BsLipA is 543 + bp.
++
+
+
+
+
Figure S4. LB Petri dishes for + constructing + IsPETase and BsLipA libraries. Morphological changes of PBAT films after + degradation.
++
+
+
+
+
Figure S5. PCR products generated by the overlap extension PCR. + The highlights in the gel tray is the POE-PCR product.
++
+
Figure S6. Hydrolysis zones of ND within 24h.
+
+
Figure S7. Morphological changes of PBAT films over different + degradation periods. After incubating for 5 days in LB liquid medium without inoculated bacteria, the + control group's PBAT remained intact, showing no significant changes. In the treatment group, PBAT films + exhibited increasing degradation rates, turning whiter in color and decreasing in ductility when + co-incubated with different strains. Films treated with IsPETase showed relatively minor changes, with a + slight reduction in overall film area and a few holes appearing on the surface. However, films treated + with BsLipA and ND strains underwent significant degradation over time, cracking into pieces, reducing + in area, and eventually degrading into almost powder form. Notably, the films treated with ND strain had + the highest degradation rate.
++
Figure S8. The three-dimensional structure of BABTaB.
+
Figure S9. The three-dimensional structure of + IsPETase.
+
Figure S10. The three-dimensional structure of ND.
+
+
Figure S11. Number of H-bonds and SASA of IsPETase and mutant ND. (a) The number of hydrogen bonds during the protein-ligand complex + simulation process;(b) The SASA curve of the protein during the protein-ligand + complex simulation process (The solvent accessible surface area (SASA) is calculated by the van der + Waals interaction between the solute and the solvent molecules. The SASA of the protein + decreases as the protein density increases, so the change of SASA can predict the change of protein + structure).
+ 
+
Figure S12. Molecular docking analysis of ND and IsPETase. (a) + Hydrogen bond interactions between the mutant ND and BABTaB. The green dashed lines represent hydrogen + bonds, with the numbers above representing the lengths of the hydrogen bonds; (b) Hydrogen bond + interactions between the IsPETase and BABTaB. The amino acid residues involved in hydrogen bond + interactions within the proteins are labeled and displayed in a stick model. The orange dashed lines + indicate hydrogen bonds, with the numbers above representing the lengths of the hydrogen bonds. +
++
+ 
Figure S13. (a) The superimposed active site clefts of
+ BABTaB-bound complex of IsPETase; (b) The superimposed active site clefts
+ of BABTaB-bound complex of ND; The
+ BABTaB-bound pocket is shown in a surface model and the BABTaB molecule is
+ shown as a stick.
+
+
+
Figure S14. Molecular dynamics trajectories of + IsPETase and ND were analyzed. IsPETase represents IsPETase and ND represents mutant ND. (a) The RMSF curve of the protein during the protein-ligand + complex simulation process (The fluctuation of each atom relative to its average position is + calculated, which characterizes the average of the structural changes over time, and gives a + characterization of the flexibility of each region of the protein. The RMSF of the ND-ligand complex has a large difference from + that of the IsPETase-ligand + complex, indicating that the active region of the protein of the ND mutant shifted); (b) The Rg curve of the protein during the + protein-ligand complex simulation process + (The radius of gyration (Rg) is used to prove the + compactness of the protein structure during the simulation process, it is the distance between the + centroid of all atoms and their ends at a specific time interval. Throughout the complex MD simulation, the fluctuation of Rg value + of IsPETase is + larger than that of ND, indicating that ND binds more + tightly to small molecules); (c) + The RMSD curve of the protein during the protein-ligand + complex simulation process + (The + complex reached equilibrium after 20 ns, indicating that the whole simulation process was stable and + reliable. The RMSD of the ND composite system showed a small decrease compared to that of the + IsPETase, + indicating that the stability of the ND composite system was higher than that of the IsPETase).
++ TABLE +
++ Table. 1S The primers which were used in + this + study.
+|
+ Number + |
+
+ Primers + |
+
+ Nucleic acid sequences(5'to3') + |
+
|
+ 1 + |
+
+ BslipA-R + |
+
+ CAGTGGTGGTGGTGGTGGTGATTCGTATTCTGTCCTCCgcc + + |
+
|
+ 2 + |
+
+ BslipA-F + |
+
+ GCAACATGTCTGCGCAGGCTGCTGAGCATAATCCAGTTGTGAT + + |
+
|
+ 3 + |
+
+ pHCM12M-SPC-F + |
+
+ GGCGGAGGACAGAATACGAATCACCACCACCACCACCACT + + |
+
|
+ 4 + |
+
+ pHCM12M-SPC-R + |
+
+ CACAACTGGATTATGCTCAGCAGCCTGCGCAGACATGTTG + + |
+
|
+ 5 + |
+
+ BslipA/IsPETase-Test-F1 + |
+
+ CCGGGACTCAGGAGCATTTAA + |
+
|
+ 6 + |
+
+ BslipA/IsPETase-Test-R1 + |
+
+ TCTAGTAGAGAGCGTTCACCGACA + |
+
|
+ 7 + |
+
+ 16sRNA-F + |
+
+ AGAGTTTGATCCTGGCTCAG + |
+
|
+ 8 + |
+
+ 16sRNA-R + |
+
+ TACGGTTACCTTGTTACGACTT + |
+
|
+ 9 + |
+
+ pHCM12M-PETase-R + |
+
+ GTCTTGAGGCGCGCGGAAAGTTAGCCTGCGCAGACATGTTG + + |
+
|
+ 10 + |
+
+ pHCM12M-PETase-F + |
+
+ GCGATTTTAGAACAGCAAATTGCTCACACCACCACCACCACCACT + + |
+
|
+ 11 + |
+
+ BslipA/PETase-F + |
+
+ CGTTAACGTTAATCTTTACGATGGCGTTCAGCAACATGTCTGCGCAGGCT + + |
+
|
+ 12 + |
+
+ BslipA/PETase-R + |
+
+ TCCGAAGATTGACTATTCTCAGATAAAGTTCAGTGGTGGTGGTGGTGGTG + + |
+
|
+ 13 + |
+
+ pHCM12M-BslipA/PETase-F + |
+
+ CACCACCACCACCACCACTGAACTTTATCT + |
+
|
+ 14 + |
+
+ pHCM12M-BslipA/PETase-R + |
+
+ AGCCTGCGCAGACATGTTGCTGAACGCCA + |
+
|
+ 15 + |
+
+ 1028-M-F1 + |
+
+ GTCCACGGTTATGGAGGGAATGATT + |
+
|
+ 16 + |
+
+ 1028-M-R1 + |
+
+ AATCATTCCCTCCATAACCGTGGAC + |
+
|
+ 17 + |
+
+ 1028-M-F2 + |
+
+ GAATGATTATAACTTCATCTCCCTTATG + |
+
|
+ 18 + |
+
+ 1028-M-R2 + |
+
+ CATAAGGGAGATGAAGTTATAATCATTCCC + |
+
|
+ 19 + |
+
+ 1028-M-F3 + |
+
+ AAGGAACGTATAGCGCAGTT + |
+
|
+ 20 + |
+
+ 1028-M-R3 + |
+
+ AACTGCGCTATACGTTCCTT + |
+
|
+ 21 + |
+
+ PETase-M-F1 + |
+
+ TTCCGGGATATAACTCGAGACAGTCATCAATTA + |
+
|
+ 22 + |
+
+ PETase-M-R1 + |
+
+ TAATTGATGACTGTCTCGAGTTATATCCCGGAA + |
+
+
Table S2. BsLipA (50μl) (Concentration system not using the (StarMut) + error-prone PCR kit)
+|
+ Reaction system(μl) + |
+
+ 1 + |
+
+ 2 + |
+
+ 3 + |
+
+ 4 + |
+
+ 5 + |
+
|
+ 2×Rapid Taq Master Mix + |
+
+ 25 + |
+
+ 25 + |
+
+ 25 + |
+
+ 25 + |
+
+ 25 + |
+
|
+ Template DNA + |
+
+ 1 + |
+
+ 1 + |
+
+ 1 + |
+
+ 1 + |
+
+ 1 + |
+
|
+ Primer 1(10 µM) + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
|
+ Primer 2(10 µM) + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
|
+ MgCl2 + |
+
+ 0.5 + |
+
+ 1 + |
+
+ 2 + |
+
+ 0.5 + |
+
+ 1 + |
+
|
+ MnCl2 + |
+
+ 0.5 + |
+
+ 0.5 + |
+
+ 0.5 + |
+
+ 1 + |
+
+ 1 + |
+
|
+ H2O + |
+
+ 18.5 + |
+
+ 18 + |
+
+ 17 + |
+
+ 17.5 + |
+
+ 17 + |
+
+
Table S3. IsPETase(50μl)
+|
+ Reaction system(μl) + |
+
+ 0 + |
+
+ 2 + |
+
+ 3 + |
+
+ 4 + |
+
+ 6 + |
+
+ 8 + |
+
+ 10 + |
+
|
+ 2×Rapid Taq Master Mix + |
+
+ 25 + |
+
+ 25 + |
+
+ 25 + |
+
+ 25 + |
+
+ 25 + |
+
+ 25 + |
+
+ 25 + |
+
|
+ Template DNA + |
+
+ 1 + |
+
+ 1 + |
+
+ 1 + |
+
+ 1 + |
+
+ 1 + |
+
+ 1 + |
+
+ 1 + |
+
|
+ Primer 1(10 µM) + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
|
+ Primer 2(10 µM) + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
|
+ Enhancer + |
+
+ 0 + |
+
+ 2 + |
+
+ 3 + |
+
+ 4 + |
+
+ 6 + |
+
+ 8 + |
+
+ 10 + |
+
|
+ H2O + |
+
+ 20 + |
+
+ 18 + |
+
+ 17 + |
+
+ 16 + |
+
+ 14 + |
+
+ 12 + |
+
+ 10 + |
+
+
+
Table S4.IsPETase(50μl)
+|
+ Reaction system(μl) + |
+
+ 1 + |
+
+ 2 + |
+
+ 3 + |
+
+ 4 + |
+
+ 5 + |
+
+ 6 + |
+
|
+ 2×Rapid Taq Master Mix + |
+
+ 25 + |
+
+ 25 + |
+
+ 25 + |
+
+ 25 + |
+
+ 25 + |
+
+ 25 + |
+
|
+ Template DNA + |
+
+ 1 + |
+
+ 1 + |
+
+ 1 + |
+
+ 1 + |
+
+ 1 + |
+
+ 1 + |
+
|
+ Primer 1(10 µM) + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
|
+ Primer 2(10 µM) + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
|
+ MgCl2 + |
+
+ 0.5 + |
+
+ 1 + |
+
+ 2 + |
+
+ 0.5 + |
+
+ 1 + |
+
+ 2 + |
+
|
+ MnCl2 + |
+
+ 1 + |
+
+ 1 + |
+
+ 1 + |
+
+ 2 + |
+
+ 2 + |
+
+ 2 + |
+
|
+ H2O + |
+
+ 18.5 + |
+
+ 18 + |
+
+ 17 + |
+
+ 17.5 + |
+
+ 17 + |
+
+ 16 + |
+
Table S5. Strains which were used in this study. +
+|
+ Names + |
+
+ Relevant + characteristic + |
+
+ Source + + |
+
|
+ SCK6 + |
+
+ Em his nprE18 aprE3 eglSΔ102 bglT/bglS EV + lacA::PxylA-comKv + |
+
+ YanXin Laboratory, Nanjing Agricultural + University + |
+
|
+ KT2440 + |
+
+ - + |
+
+ YanXin Laboratory, Nanjing Agricultural + University + |
+
|
+ DH5α + |
+
+ deoR endA1 gyrA96 hsdR17 (rk-mk+)recA1 relA1 supE44 + thi- 1 Δ(lacZYA-argF)U169 Φ80lacZ ΔM15F - λ - + |
+
+ Vazyme + |
+
SCK6 Accession Number: The ultra-competent Bacillus subtilis strain SCK6 can be obtained from the Bacillus Genetic Stock + Center (http://www.bgsc.org), with the accession number 1A976; KT2440 Accession Number: txid160488; + pBBR-tph Accession Number: txid101510.
++ + + +
| Number | +Name | +Nucleic acid sequences (5' to 3') | +
|---|---|---|
| 1 | +The signal peptide sequence for extracellular expression in Bacillus subtilis |
+ ATGAGAAGTAAAAAATTGTGGATAAGT TTGTGTTTGCGTTAACGTTAATCTTTAC GATGGCGTTCAGCAACATGTCTGCGCAG GCT + |
+
| 2 | +The signal peptide sequence for expression in KT2440 | +ATGAAATACCTGCTGCCGACCCTGCTG CTGGTCTGCTCCTCCTCGCTGCCCAGCC GGCGATGGCC |
+
| 3 | +BsLipA | +GCTGAGCATAATCCAGTTGTGATGGTG CACGGCATTGGTGGTGCCTCTTATAACT TTTTTTCTATTAAAAATTACTTGATTGG ACAAGGCTGGGATCGAAACCAATTATAT GCTATTGATTTCATAGACAAAACAGGAA ATAACCGCAACAATGGTCCGCGTCTATC GAGATTCGTCAAAGATGTGTTAGACAAA ACGGGTGCCAAAAAAGTAGATATTGTGG CTCATAGTATGGGCGGGGCGAACACGCT ATATTATATTAAGAATCTAGATGGCGGC GATAAAATTGAGAACGTTGTCACAATTG GTGGAGCAAACGGACTCGTTTCAAGCAG AGCATTACCAGGCACAGATCCAAATCAA AAAATTCTTTACACATCCGTCTATAGCT CAGCAGATCTTATTGTCGTCAACAGCCT CTCTCGTTTAATTGGCGCAAGAAACATC CTGATCCATGGCGTTGGCCATATCGGTC TATTAACCTCAAGCCAAGTGAAAGGGTAT ATTAAAGAAGGACTGAACGGCGGAGGAC AGAATACGAAT + |
+
| 4 | +IsPETase | +CAAACGAACCCGTATGCGAGAGGCCCTA AATCCGACAGCCGCATCTCTTGAAGCAT CAGCAGGCCCGTTTACAGTTCGTTCTTT TACAGTTTCAAGACCGTCAGGATATGGA GCAGGAACAGTTTATTATCCGACAAATG CAGGCGGCACAGTTGGAGCAATTGCTAT TGTTCCGGGATATACAGCGAGACAGTCA TCAATTAAATGGTGGGGACCGAGATTGG CAAGCCATGGATTTGTTGTTATTACAAT TGATACGAACAGCACACTGGATCAACCG TCATCAAGATCTTCACAACAAATGGCAG CACTTCGTCAGGTTGCAAGCCTGAACGG CACGTCATCATCACCGATTTATGGCAAA GTTGATACAGCAAGAATGGGCGTCATGG GCTGGTCAATGGGCGGAGGAGGATCACT GATTTCAGCAGCAAATAATCCGTCACTT AAAGCAGCAGCGCCGCAAGCACCGTGGA TTCATCAACGAATTTTTCATCAGTTACA GTTCCGACGCTGATCTTTGCATGCGAAA ACGATAGCATTGCACCGGTGAACAGCAG CGCCCTGCCTATTTATGATTCAATGTCA AGAAATGCGAAACAATTTCTGGAAATTA ACGGCGGCTCACATTCTTGCGCAAATAG CGGAAATTCAAATCAGGCACTTATTGGC AAGAAGGGCGTTGCATGGATGAAAAGAT TTATGGATAATGATACGCGCTATAGCAC ATTTGCATGTGAAAATCCTAATAGCACG AGAGTGAGCGATTTTAGAACAGCAAATT GCTCA + |
+
| 5 | +ND | +GCTAACCCTTATGAAAGAGGACCTAACC CTACAGATGCATTACTGGAAGCTCGCAG CGGACCTTTTTCAGTTTCAGAAGAGAAT GTGTCTAGACTTAGCGCTAGCGGATTTG GAGGCGGAACAATCTACTATCCTAGAGA AAACAATACGTACGGAGCAGTGGCAATT TCTCCTGGATATACGGGAACAGAAGCTA GCATTGCATGGCTTGGCGAACGCATTGC CTCTCACGGATTTGTTGTCATTACAATT GATACAATTACGACACTTGATCAACCGG ACTCTAGAGCCGAACAGTTAAATGCTGC ATTAAACCATATGATTAACAGAGCCTCA TCTACAGTGAGATCAAGAATCGATTCAT CTAGATTAGCAGTCATGGGACATAGCAT GGGAGGAGGAGGCTCTCTTCGCTTAGCT TCACAAAGACCGGACCTGAAAGCAGCAA TTCCTTTAACGCCGTGGCATCTGAACAA AACTGGAGCTCAGTGACAGTTCCGACGT TAATCATTGGCGCTGATTTAGATACAAT TGCCCCGGTCGCAACATCTGCCAAGCCT ATCTATAACTCTTTACCTAGCAGCATTA GCAAGGCATATCTTGAATTAGATGGAGC CACACATTTTGCGCCGAACATTCCTAACA AGATCATTGGCAAATATTCAGTAGCTTG GCTTAAGAGATTCGTCGACAATGACACA CGCTATACACAATTTCTTTGTCCGGGAC CGAGAGATGGATTATTTGGCGAAGTTGA AGAATACAGATCAACATGTCCTTTT + |
+
| 6 | +The result of fragment sequencing under the error-prone PCR system added 0 μL StarMut Enhancer | +CAAACGAACCCGTATGCGAGAGGCCCTA AATCCGACAGCCGCATCTCTTGAAGCAT CAGCAGGCCCGTTTACAGTTCGTTCTTT TACAGTTTCAAGACCGTCAGGATATGGA GCAGGAACAGTTTATTATCCGACAAATG CAGGCGGCACAGTTGGAGCAATTGCTAT TGTTCCGGGATATACAGCGAGACAGTCA TCAATTAAATGGTGGGGACCGAGATTGG CAAGCCATGGATTTGTTGTTATTACAAT TGATACGAACAGCACACTGGATCAACCG TCATCAAGATCTTCACAACAAATGGCAG CACTTCGTCAGGTTGCAAGCCTGAACGG CACGTCATCATCACCGATTTATGGCAAA GTTGATACAGCAAGAATGGGCGTCATGG GCTGGTCAATGGGCGGAGGAGGATCACT GATTTCAGCAGCAAATAATCCGTCACTT AAAGCAGCAGCGCCGCAAGCACCGTGGA TTCATCAACGAATTTTTCATCAGTTACA GTTCCGACGCTGATCTTTGCATGCGAAA ACGATAGCATTGCACCGGTGAACAGCAG CGCCCTGCCTATTTATGATTCAATGTCA AGAAATGCGAAACAATTTCTGGAAATTA ACGGCGGCTCACATTCTTGCGCAAATAG CGGAAATTCAAATCAGGCACTTATTGGC AAGAAGGGCGTTGCATGGATGAAAAGAT TTATGGATAATGATACGCGCTATAGCAC ATTTGCATGTGAAAATCCTAATAGCACG AGAGTGAGCGATTTTAGAACAGCAAATT GCTCA + |
+
| 7 | +The result of fragment sequencing under the error-prone PCR system added 4 μL StarMut Enhancer | +GATCACTGATTTGACGACGAAATAATCC GTCACTTAAAGCAGCAGCGCCGAAGC ACCGTGGGATTCATCAACGAATTTTCA TCAGTTACAGTTCCGAGCGTACTTTG CATGCGAACGGAACGATAGCATGCGC TGAACAGCAGCGCCGGCCTTATTATG TCAATGTCAAGAAATGGCGCGAACAA TTCTGGAAATTAACGCGGCGCTCAATTCT TGCAGCAATAGCGGAAATTCAATCAG GCACTTATTGGCAAGAAGGGCGTTGCA TGGATGAAAAGATTTATGGATAATGATG CGGCTATAGCACATTTGCATGTGAAAA TCCCTAATAGCACGAGAGTGAGCGATT AGAACAGCAAAATTGTCTCA + |
+
| 8 | +The result of fragment sequencing under the error-prone PCR system added 6 μL StarMut Enhancer | +CAAACGAACCCGTATGCGAGAGGCCCT AATCCGACAGCCGCATCTCTTGAAGCA TCAGCAGGCCCGTTACAGTTCGTTCTTT TTACAGTTTCAAGACCGTCAGGATATGG AGCAGGAACAGTTTATTATCCGACAAAT GCAGGCGGCACAGTTGGAGCAATTGCT ATTGTTCCGGGATATACAGCGAGACAGT CATCAATTAAATGGTGGTGGGACCGAGA TTGGCAAGCCATGGATTTGTTGTTATTAC ATTGATACGAACAGCACACTGGATCAAC CGTCATCAAGATCTTCACAACAAATGG CACTTCGTCAGGTTGCAAGCCTGAACG CACGTCATCATCACCGATTTATGGCAAA GTTGATACAGCAAGAATGGGCGTCATGG GCTGGTCAATGGGCGGAGGAGGATCACT GATTTCAGCAGCAAATAATCCGTCACTT AAAGCAGCAGCGCCGCAAGCACCGTGG ATTCATCAACGAATTTTTCATCAGTTAC GTTCCGACGCTGATCTTTGCATGCGAAA ACGATAGCATTGCACCGGTGAACAGCAG CGCCCTGCCTATTTATGATTCAATGTCA AGAAATGCGAAACAATTTCTGGAAATTA ACGGCGGCTCACATTCTTGCGCAAATAG CGGAAATTCAAATCAGGCACTTATTGGC AAGAAGGGCGTTGCATGGATGAAAAGAT TTATGGATAATGATACGCGCTATAGCAC ATTTGCATGTGAAAATCCTAATAGCACG AGAGTGAGCGATTTTAGAACAGCAAATT GCTCA + |
+
| 9 | +The result of fragment sequencing under the error-prone PCR system added 8 μL StarMut Enhancer | +CAAACGAACCCGTATGCGAGAGGCCCT AATCCGACAGCCGCATCTCTTGAAGCA TCAGCAGGCCCGTTACAGTTCGTTCTTT TTACAGTTTCAAGACCGTCAGGATATGG AGCAGGAACAGTTTATTATCCGACAAAT GCAGGCGGCACAGTTGGAGCAATTGCT ATTGTTCCGGGATATACAGCGAGACAGT CATCAATTAAATGGTGGTGGGACCGAGA TTGGCAAGCCATGGATTTGTTGTTATTAC ATTGATACGAACAGCACACTGGATCAAC CGTCATCAAGATCTTCACAACAAATGG CACTTCGTCAGGTTGCAAGCCTGAACG CACGTCATCATCACCGATTTATGGCAAA GTTGATACAGCAAGAATGGGCGTCATGG GCTGGTCAATGGGCGGAGGAGGATCACT GATTTCAGCAGCAAATAATCCGTCACTT AAAGCAGCAGCGCCGCAAGCACCGTGG ATTCATCAACGAATTTTTCATCAGTTAC GTTCCGACGCTGATCTTTGCATGCGAAA ACGATAGCATTGCACCGGTGAACAGCAG CGCCCTGCCTATTTATGATTCAATGTCA AGAAATGCGAAACAATTTCTGGAAATTA ACGGCGGCTCACATTCTTGCGCAAATAG CGGAAATTCAAATCAGGCACTTATTGGC AAGAAGGGCGTTGCATGGATGAAAAGAT TTATGGATAATGATACGCGCTATAGCAC ATTTGCATGTGAAAATCCTAATAGCACG AGAGTGAGCGATTTTAGAACAGCAAATT GCTCA + |
+
| 10 | +The result of fragment sequencing under the error-prone PCR system added 10 μL StarMut Enhancer | +TCTGCTGCTTAGGAGATTTTCAGATGCA AATGTGCTATAGGCGCGTATCATTATCC AAATCTTTTCATCCATGACCAGCGCCTT TTGGCAAATAGTGCGCGATGGAATTTGC CGCTATTTGCCGAAGATTTGCTGGTCAA CGTTAATTCCGAAGATTGTGTGTGAGGG CTGACATTGAATCATAAATAGGGCAGGG CGGCTGTGTTTCACCGGTGCAATGCTAT CGTTTTCGCATGCAAAGATCAGGCTGGA ACTGTAACCTGATGAAAATGTGCTGGTG ATGAATCCCGGGTCGTTGGGGCTGGTGA CTGCTTAAGTGACCGGATTATTTGCTGC TGAACTAGTGATCCCTCCTCCGCATTTC GACCAGCCCAGGACGCCCATCTGCTGCA GTATCACGTTGCAATAAGGCTGGTGATG ATGAGTCCGTTGCTAGGTTGGCAGTGCA GACGAAGTGCCTCATTGTTGTGTCGAAA ATCTTGATGACGGTGTCAGTTGGTGTCT GTTGTGATCAATTGTAATAACAACAAAT CCATGGCCTGCCAATCTGCGGCTCCACC ATTTAGTGATGACTGCTGCTTGTCCAAT CCCGAACCAATAGCAATTGCTCCGAACT GTGCCGCTCGCATTGTGCGATAAACTGA CTGTTCGTGCTCCATATCCTGGACGTCT TGAACTGTAAAAGAACTGGTGACGTAAA CGGGCTGTGATGATGGCTGAGGATGACG GGGTTCGGTGGTGATGGTGCTGGTGGCG + |
+
| 11 | +The result of fragment sequencing under the error-prone PCR system added 0.5 μL Mg2+ and + 1 μL + Mn2+ (without using StarMut Error-Prone PCR Kit) | +CTAACTGCCATCAGGCGCGCCTAAAA CAAACGAACCCGTATGCGAGAGGCCCT AATCCGACAGCCGCATCTCTTGAAGCA TCTGCAGGCCCGTTACAGTTCGTTCTCT TTACAGTTTCAAGACCGTCAGGATATGG AGCAGGAACAGTTTATTATCCGACAAAT GTAGGCGGCACAGTTGGAGCAATTGCT ATTGTTCCGGGATATACAGCGAGACAGT CATCAATTAAATGGTGGTGGGACCGAGA TTGGCAAGCCATGGATTTGTTGTTATTAC ATTGATACGAACAGCACACTGGATCAAC CGTCATCAAGATCTTCACAACAAATGG CACTTCGTCAGGTTGCAAGCCTGAACG CACGTCATCATCACCGATTTATGGCAAA GTTGATACAGCAAGAATGGGCGTCATGG GCTGGTCAATGGGCGGAGGAGGATCACT GATTTCAGCAGCAAATAATCCGTCACTT AAAGCAGCAGCGCCGCAAGCACCGTGG ATTCATCAACGAATTTTTCATCAGTTAC GTTCCGACGCTGATCTTTGCATGCGAAA ACGATAGCATTGCACCGGTGAACAGCAG CGCCCTGCCTATTTATGATTCAATGTCA AGAAATGCGAAACAATTTCTGGAAATTA ACGGCGGCTCACATTCTTGCGCAAATAG CGGAAATTCAAATCAGGCACTTATTGGC AAGAAGGGCGTTGCATGGATGAAAAGAT TTATGGATAATGATACGCGCTATAGCAC ATTTGCATGTGAAAATCCTAATAGCACG AGAGTGAGCGATTTTAGAACAGCAAATT GCTCA + |
+
| 12 | +The amino acid sequence of IsPETase | +
+ MNFPRASRLMQAAVLGGLMAVSAAATA + QTNPYARGPNPTAASLEASAGPFTVRSFT + VSRPSGYGAGTVVYPTNAGGTVGAIAIV + PGYTARQSSIKWWGPRLASGHFVVITIDT + NSTLDQPSSRSSQQMAALRQVASLNGTS + SSPJYGKVDTARMGVMGWMSGGGGSLLI + SAANNPSLKAAAPQAPWDSSNTFSSVTV + PTLIFACENDSIAPVNSSALPIYDSMSRNA + KQFLEINGGSHSCANSGNSNQALIGKKG + VAWMKRFMDNDTRYSTFACENPNSTRVS + DFRTANCS + |
+
| 13 | +The amino acid sequence of TfCut | +
+ MNFPRASRLMQAAVLGGLMAVSAAATA + QTNPYARGPNPTAASLEASAGPFTVRSFT + VSRPSGYGAGTVVYPTNAGGTVGAIAIV + PGYTARQSSIKWWGPRLASGHFVVITIDT + NSTLDQPSSRSSQQMAALRQVASLNGTS + SSPJYGKVDTARMGVMGWMSGGGGSLLI + SAANNPSLKAAAPQAPWDSSNTFSSVTV + PTLIFACENDSIAPVNSSALPIYDSMSRNA + KQFLEINGGSHSCANSGNSNQALIGKKG + VAWMKRFMDNDTRYSTFACENPNSTRVS + DFRTANCS + |
+
| 14 | +The amino acid sequence of ND | +
+ ANPYERGPNPTDALLEARSGPFVSEEN + VSRLSASGFGGGTIYYPRENNTYGAVAIS + PGYTGEASIAWLGERIASGHFVVITIDTI + TTLDQPDRAEQLNAALNHMINRASSTTV + RSRIDSSRRLAVMGHSMGGGGGSLRLASQR + PDLKAAIPLTPWHLNKNWSSSVTVPTLIIG + ADLDTIAPVATSIAKPIYNSSLPSSISKAYLE + LDGATHFAPNIPNKIIGKYSVAWLKRFVD + NDTRYTQFLCPGPRDGFLGVEEEYRSRTC + PF + |
+