Coding

Part:BBa_M50054:Design

Designed by: Lucy Maynard, Doris Mai and Amy Weissenbach   Group: Stanford BIOE44 - S11   (2016-12-11)
Revision as of 07:23, 12 December 2016 by DorisMai (Talk | contribs)


PETase double mutant I208V and R90A


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 633
    Illegal PstI site found at 864
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 633
    Illegal PstI site found at 864
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 633
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 633
    Illegal PstI site found at 864
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 633
    Illegal PstI site found at 864
    Illegal NgoMIV site found at 220
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 388


Design Notes

Since each individual mutant (I208V and R90A) showed imrpovement on PETase catalytic activity, our design thinking is based on the hypothesis that the combination of the two mutations will further enhance the PETase catalytic activity. 6 His tag is also appended at the end of the PETase sequence for western blotting test in case we want to verify whether the protein is being successfully made or not. The entire sequence is optimized for Escherichia coli by IDT’s codon optimization tool to imrpove the expression of PETase gene since it is not from E.coli.

Source

Wild type PETase sequence is from Ideonella sakaiensis and is identified by Yoshida et al. [1]. Two mutation sites are originally designed by 2016 iGEM Tianjin team [2].

References

[1] Yoshida, Shosuke, et al. 2016. "A bacterium that degrades and assimilates poly (ethylene terephthalate)." Science 351.6278: 1196-1199.

[2] IGEM16_Tianjin. "Part:BBa_K2110105." Part:BBa K2110105. IGEM, 12 Oct. 2016. Web. 11 Dec. 2016.