Difference between revisions of "Part:BBa K2110800"
 
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This codon optimized sequence codes the wild type protein called PETase. PETase is a Poly(ethylene terephthalate) hydrolase, which was found in Ideonella sakaiensis (strain 201-F6) by Japanese scientists. It allows Ideonella sakaiensis to degrade PET. If properly transferred into engineered organisms as E.Coli or yeast, secreted PETase can catalyze the hydrolysis of PET to produce mono(2-hydroxyethyl) terephthalate (MHET) as the major product. Optimum temperature is 40 degrees Celsius for PET film hydrolysis and optimum pH is 9.
 
This codon optimized sequence codes the wild type protein called PETase. PETase is a Poly(ethylene terephthalate) hydrolase, which was found in Ideonella sakaiensis (strain 201-F6) by Japanese scientists. It allows Ideonella sakaiensis to degrade PET. If properly transferred into engineered organisms as E.Coli or yeast, secreted PETase can catalyze the hydrolysis of PET to produce mono(2-hydroxyethyl) terephthalate (MHET) as the major product. Optimum temperature is 40 degrees Celsius for PET film hydrolysis and optimum pH is 9.
  
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===Usage and Biology===
 
===Usage and Biology===
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https://static.igem.org/mediawiki/parts/thumb/4/44/Prediction.png/569px-Prediction.png
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https://static.igem.org/mediawiki/parts/5/5a/SecretionofPETase.jpeg
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In order to build a PET degradation device, we had to increase the secretion of PET degradation enzymes. So we searched for a novel signal peptide candidate through literature search and determined the cleavage site using computational analysis.
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<p>In order to release PETase (BBa_K2110800) from <em>E. coli</em>, the endogenous signal peptide was removed and a new signal peptide (NSP4) was attached. As a result, it can be seen that NSP4-PETase was secreted more into the media than PETase (BBa_K2110800).</p>
  
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===Sequence and Features===
 
===Sequence and Features===
 
<partinfo>BBa_K2110800 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2110800 SequenceAndFeatures</partinfo>
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Activity Assay<br/>
 
Activity Assay<br/>
 
We detect the absorption peak of culture medium after culturing with PET film added for 2d. The absorption degree-wavelength curve is as follows.
 
We detect the absorption peak of culture medium after culturing with PET film added for 2d. The absorption degree-wavelength curve is as follows.
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https://static.igem.org/mediawiki/parts/6/60/T--Tianjin--partwt.png
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https://static.igem.org/mediawiki/parts/thumb/6/60/T--Tianjin--partwt.png/800px-T--Tianjin--partwt.png
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===References===
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Han, S., Machhi, S., Berge, M., Xi, G., Linke, T., & Schoner, R. (2017). Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxin H35L in Escherichia coli. AMB Express, 7(1), 93.
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 22:24, 21 October 2019


PETase

This codon optimized sequence codes the wild type protein called PETase. PETase is a Poly(ethylene terephthalate) hydrolase, which was found in Ideonella sakaiensis (strain 201-F6) by Japanese scientists. It allows Ideonella sakaiensis to degrade PET. If properly transferred into engineered organisms as E.Coli or yeast, secreted PETase can catalyze the hydrolysis of PET to produce mono(2-hydroxyethyl) terephthalate (MHET) as the major product. Optimum temperature is 40 degrees Celsius for PET film hydrolysis and optimum pH is 9.


Usage and Biology

569px-Prediction.png

SecretionofPETase.jpeg

In order to build a PET degradation device, we had to increase the secretion of PET degradation enzymes. So we searched for a novel signal peptide candidate through literature search and determined the cleavage site using computational analysis.

In order to release PETase (BBa_K2110800) from E. coli, the endogenous signal peptide was removed and a new signal peptide (NSP4) was attached. As a result, it can be seen that NSP4-PETase was secreted more into the media than PETase (BBa_K2110800).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 79
    Illegal BglII site found at 289
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Activity Assay
We detect the absorption peak of culture medium after culturing with PET film added for 2d. The absorption degree-wavelength curve is as follows. 800px-T--Tianjin--partwt.png

References

Han, S., Machhi, S., Berge, M., Xi, G., Linke, T., & Schoner, R. (2017). Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxin H35L in Escherichia coli. AMB Express, 7(1), 93.