Difference between revisions of "Part:BBa K2910003"
 
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This part encodes ''Ideonella sakaiensis'' poly(ethylene terephthalate) hydrolase with several mutations predicted to increase its catalytic activity. The bulky arginine residue at 280 has been mutated to an alanine to reduce steric clash with PET. The other mutations are predicted to increase interaction between isPETase and aromatic moieties of PET.
 
This part encodes ''Ideonella sakaiensis'' poly(ethylene terephthalate) hydrolase with several mutations predicted to increase its catalytic activity. The bulky arginine residue at 280 has been mutated to an alanine to reduce steric clash with PET. The other mutations are predicted to increase interaction between isPETase and aromatic moieties of PET.
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Internal working name: Dimi1
  
 
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===Usage and Biology===
 
===Usage and Biology===
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<div style="text-align: center;">
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<partinfo>BBa_K2910003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2910003 SequenceAndFeatures</partinfo>
  
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In order to assess the enzymatic activity of PETase variants relative to the wild type, and W159H S238F PETase (BBa_k2910000), p-nitrophenyl butyrate assays were performed. Ester bonds in PET plastic are mimicked by p-nitrophenol. Each PETase variant cleaves the ester bond; once the bond is broken it emits a wavelength at 405 nm. This emission can be read by a plate reader; if PETase is catalytically active, absorbance at 405 should be perceived. The following figures show the results of the aforementioned assay for this variant.
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A plot of pNB hydrolysis by BBa_K2910003 at pH = 8, concentration = 2500uM; is shown below:
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<img scr="https://static.igem.org/mediawiki/parts/0/06/T--Toronto--pNPB-Hydrolysis-pH-8_Dimi-2500uM.png">
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A plot of pNB hydrolysis by BBa_K2910003 at pH = 8, concentration = 625 uM; is shown below:
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<img scr="https://static.igem.org/mediawiki/parts/c/ca/T--Toronto--pNPB-Hydrolysis-pH-8_Dimi-625uM.png">
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A plot of pNB hydrolysis by BBa_K2910003 at pH = 8, concentration = 156uM; is shown below:
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<img scr="https://static.igem.org/mediawiki/parts/7/7d/T--Toronto--pNPB-Hydrolysis-pH-8_Dimi-156uM.png">
  
 
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Latest revision as of 03:57, 22 October 2019


IsPETase-T88F-I208Y-S238F-S245Y-R280A with C-terminal Hexahistidine Tag

This part encodes Ideonella sakaiensis poly(ethylene terephthalate) hydrolase with several mutations predicted to increase its catalytic activity. The bulky arginine residue at 280 has been mutated to an alanine to reduce steric clash with PET. The other mutations are predicted to increase interaction between isPETase and aromatic moieties of PET.

Internal working name: Dimi1

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]

In order to assess the enzymatic activity of PETase variants relative to the wild type, and W159H S238F PETase (BBa_k2910000), p-nitrophenyl butyrate assays were performed. Ester bonds in PET plastic are mimicked by p-nitrophenol. Each PETase variant cleaves the ester bond; once the bond is broken it emits a wavelength at 405 nm. This emission can be read by a plate reader; if PETase is catalytically active, absorbance at 405 should be perceived. The following figures show the results of the aforementioned assay for this variant.

A plot of pNB hydrolysis by BBa_K2910003 at pH = 8, concentration = 2500uM; is shown below:


<img scr="T--Toronto--pNPB-Hydrolysis-pH-8_Dimi-2500uM.png">


A plot of pNB hydrolysis by BBa_K2910003 at pH = 8, concentration = 625 uM; is shown below:

<img scr="T--Toronto--pNPB-Hydrolysis-pH-8_Dimi-625uM.png">


A plot of pNB hydrolysis by BBa_K2910003 at pH = 8, concentration = 156uM; is shown below:

<img scr="T--Toronto--pNPB-Hydrolysis-pH-8_Dimi-156uM.png">