Part:BBa_K3288013

signal peptide (NSP4) fused PETase(28-290;S121E/D186H/R280A)

This is a basic part that was synthesized form IDT.

NSP4-PETase is codon optimized for E. coli K12.

This PETase has three amino acids mutated (S121E/D186H/R280A).

This mutant is heat stable and also has a higher enzyme activity on PET compared to WT.

Usage and Biology

After PETase and MHETase was analyzed based on protein structures, some of them were reported to be point mutated with increased enzyme activation. Our team checked PETase wild type, W159H/S238F, and S121E/D186H/R280A’s PET degradation activities to choose enzymes for PET degrading device. These enzymes were produced at 18 °C in LB (buffered 50mM BTP (pH8.5) for 24 hours. After centrifuged, we put PET disc into cleared media with no E.coli and reacted it for 24 hours at 40 degrees Celsius. Everyday, we replaced media with ones that has new PET degrading enzyme secreted. After 5 days, we washed PET disc and dried it up to measure the change in weight. Results showed that NSP4-PETase (S121E/D186H/R280A) degraded the most PET.

Sequence and Feature

Assembly Compatibility:

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

  Illegal XhoI site found at 850
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 22
  
• 1000
  COMPATIBLE WITH RFC[1000]