Difference between revisions of "Part:BBa M50054:Design"

Latest revision as of 07:30, 12 December 2016

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 improvement 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 improve 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.

@@ Line 11: / Line 11: @@
 ===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].
+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===