Difference between revisions of "Part:BBa K2010999"

Revision as of 00:44, 22 October 2019

PETase (PET-degrading enzyme, origin I. sakaiensis)

PETase is a poly(ethylene terehphthalate)-degrading enzyme first identified in Ideonella sakaiensis. BBa_K2010999 is the sequence for the E. coli K12 optimized DNA sequence for PETase. The catalytic activity of IsPETase can be characterized using a p-nitrophenol butyrate (pNPB) assay, in which PETase cleaves the ester bond of pNPB to release p-nitrophenolate. The absorbance of this compound can be measured at 405 nm to reflect IsPETase activity.

T--Toronto--WT.SDSPAGE.png

This SDS-PAGE gel demonstrates successful protein purification of PETase using His tag affinity purification. PETase has a molecular weight of approximately 30 kDa.

This graph demonstrates PETase activity with various substrate concentrations at a pH of 7.0.

This graph demonstrates PETase activity with various substrate concentrations at a pH of 8.0.

This graph demonstrates PETase activity with various substrate concentrations at a pH of 9.0.

This graph demonstrates PETase activity as a function of pH. PETase is most active at a pH of 9.0, but activity at 8.0 and 9.0 is practically indistinguishable.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NotI site found at 67
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 9: / Line 9: @@
 https://2019.igem.org/wiki/images/9/92/T--Toronto--WT.SDSPAGE.png
-An SDS-PAGE gel demonstrates successful protein purification of PETase, which has a molecular weight of approximately 30 kDa.
+This SDS-PAGE gel demonstrates successful protein purification of PETase using His tag affinity purification. PETase has a molecular weight of approximately 30 kDa.
 https://static.igem.org/mediawiki/parts/a/a7/T--Toronto--pNPB-Hydrolysis-pH-7_Wildtype-All-Conc.png
@@ Line 25: / Line 25: @@
 https://static.igem.org/mediawiki/parts/5/54/T--Toronto--pNPB-Hydrolysis-Wildtype_pH7-8-9.png
-This graph demonstrates PETase activity as a function of pH. PETase is most active at a pH of 9.0.
+This graph demonstrates PETase activity as a function of pH. PETase is most active at a pH of 9.0, but activity at 8.0 and 9.0 is practically indistinguishable.
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