Part:BBa_K3288058

light inducible expression of signal peptide (NSP4) fused PETase(28-290;S121E/D186H/R280A)

BBa_K3288058 is a composite part in which BBa_K1075044, BBa_K3288007 and BBa_K3288013 are combined.

This part is compatible with RFC10.

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 BglII site found at 2171
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 63
  Illegal NgoMIV site found at 195
  Illegal NgoMIV site found at 289
  Illegal NgoMIV site found at 582
  Illegal NgoMIV site found at 1076
  Illegal NgoMIV site found at 1094
  Illegal NgoMIV site found at 1184
  Illegal AgeI site found at 414
  Illegal AgeI site found at 1542
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1643
  Illegal BsaI.rc site found at 525