Difference between revisions of "Part:BBa K2910000"
 
(12 intermediate revisions by the same user not shown)
Line 7: Line 7:
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
−
https://2019.igem.org/wiki/images/5/53/T--Toronto--Improv.png
+
https://2019.igem.org/wiki/images/5/53/T--Toronto--Improv.png  
  
 
<div style="text-align: center;">
 
<div style="text-align: center;">
−
https://static.igem.org/mediawiki/parts/c/cd/T--Toronto--pNPB-Hydrolysis-pH-9_All-PETase-Mutants.png
 
  
−
https://static.igem.org/mediawiki/parts/c/cd/T--Toronto--pNPB-Hydrolysis-pH-9_All-PETase-Mutants.png
+
 
  
 
</div>
 
</div>
Line 19: Line 18:
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2910000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2910000 SequenceAndFeatures</partinfo>
 +
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.
 +
 +
<img src="https://2019.igem.org/wiki/images/5/53/T--Toronto--Improv.png">
 +
<img src="https://2019.igem.org/wiki/images/e/eb/T--Toronto--AddgeneWTandTinayu.png">
  
  

Latest revision as of 03:59, 22 October 2019


IsPETase-W159H-S238F with C-terminal Hexahistidine Tag

BBa_K2910000 encodes an alpha/beta-hydrolase that degrades poly(ethylene terephthalate) (PET) into mono(2-hydroxyethyl) terephthalic acid (MHET) with trace amounts of terephthalic acid (TPA), and bis(2-hydroxyethyl)-TPA (BHET). The enzyme originates from Ideonella sakaiensis, a microbe that was discovered to assimilate PET plastic in 2016 by Yoshida et al. This part has been codon-optimized for E. coli K-12 and contains a C-terminal hexahistidine tag for affinity protein purification. Additionally, the part contains two mutations that result in the nonsynonymous amino acid substitutions W159H and S238F relative to the wildtype sequence. These mutations enhance the catalytic activity of the enzyme by narrowing the active site, which makes PETase more "cutinase-like" according to X-ray crystallography structure analysis by Austin et al (2018).

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.

<img src="T--Toronto--Improv.png"> <img src="T--Toronto--AddgeneWTandTinayu.png">