Part:BBa_K5094006

T7-IsPETase (BBa_K5094000-BBa_K5094002)

After amplifying the wild type of IsPETase gene via PCR and using enzyme digestion strategy to clone it to the downstream of the T7 promoter to make a composite part, BBa_K5094006, used to make several mutations on IsPETase gene via site-directed mutagenesis and was transformed into DE3BL21 bacteria for functional assays.

Fig 2: After cloning IsPETase downstream of the T7 promoter plasmid, bacterial transformation was done, and bacterial colonies were grown on the LB-Amp selection plates. The colonies were directly inoculated to perform the PCR technique via the IsPETase primer set. In Fig2, the 1% agarose gel showed that bacterial colonies # 1, 2, and 5 had a distinct IsPETase single PCR product band, which indicated that the bacterial colonies contained our team’s composite part, BBa_K5094006 (T7-IsPETase).

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-IsPETaseThr116Ala, lane 4 was T7-IsPETaseThr116Ala/M154Thr,and lane 5 was T7-IsPETaseThr116Ala/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-IsPETaseThr116A (-)IPTG, and lane 5 is T7-IsPETaseThr116A (+0.5mM)IPTG. Lane 6 is T7-IsPETaseThr116A/k259Glu (-)IPTG, and lane 7 is T7-IsPETaseThr116A/k259Glu (+0.5mM)IPTG. Lane 8 is T7-IsPETaseThr116A/M154Thr (-)IPTG, and lane 9 is T7-IsPETaseThr116A/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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 245
  
• 1000
  COMPATIBLE WITH RFC[1000]