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T7-IsPETase^{Thr116Ala/Met154Thr} (BBa_K5094000-BBa_K5094004): After using BBa_K5094007, as a DNA template to do site-directed mutagenesis and the team designed the 2nd mutation on the forward and the reverse primers, originally switching amino acid 186 from aspartic acid to histidine (D186H). PCR was performed, parental DNA template was digested by DpnI and transformed into bacteria. However, the IsPETase gene sequence included multiple DNA fragments with repeat regions that our team didn’t anticipate during the design of the mutations, which led to challenges in using the site-directed mutagenesis technique. The primers, which were intended to switch specific amino acids by introducing mutant nucleotides, ended up targeting incorrect amino acids due to these repeat regions. Our team created T7-IsPETase^{Thr116Ala/Met154Thr} instead.

IsPETase^{Thr116Ala/Met154Thr}, double mutations on the amino acids 116 and 154 in the IsPETase gene. The amino acid 116 from the polar uncharged side chain of the Threonine switched to the hydrophobic side chain of the alanine, and amino acid 154 from the hydrophobic side chain of the methionine to the polar uncharged side chain of the Threonine, changing the properties of the amino acids 116 and 154, altering the interaction with the substrate or the catalytic function.

Fig5: After sequencing, the mutations of the IsPETase the team created: lane 1 was a 1kb DNA ladder, and lane 2 was T7-IsPETase, lane 3 was T7-IsPETase^Thr116Ala, lane 4 was T7-IsPETase^{Thr116Ala/M154Thr}, and lane 5 was T7-IsPETase^{Thr116Ala/K259Glu}.

Fig 8: The 18% SDS-PAGE gel showed the team successfully extracted the whole protectin extract. Lane 1 is a protein-size ladder. Lane 2 is T7-IsPETase (-)IPTG,and lane 3 is T7-IsPETase (+0.5mM)IPTG.Lane 4 is T7-IsPETase^Thr116A (-)IPTG, and lane 5 is T7-IsPETase^Thr116A (+0.5mM)IPTG. Lane 6 is T7-IsPETase^{Thr116A/k259Glu} (-)IPTG, and lane 7 is T7-IsPETase^{Thr116A/k259Glu} (+0.5mM)IPTG. Lane 8 is T7-IsPETase^{Thr116A/M154Thr} (-)IPTG, and lane 9 is T7-IsPETase^{Thr116A/M154Thr} (+0.5mM)IPTG. IsPETase protein has about 30 kDa molecular weight based on the Harvard 2016 team’s part information.

Fig 9a: The co-cultured experiment data didn’t show any clear patterns of the TPA product increasing in the DE3 bacteria containing T7-IsPETase along with several IsPETase mutants, respectively, in the presence of the IPTG during the time course experiment. For the experimental controls, DE3 bacteria only, along with several IsPETase mutants that lack IPTG still also showed TPA product generation.

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