Difference between revisions of "Part:BBa K2110800"

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===Usage and Biology===
 
===Usage and Biology===
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Secretion of PETase in E. coli
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https://static.igem.org/mediawiki/parts/thumb/4/44/Prediction.png/569px-Prediction.png
  
 
https://static.igem.org/mediawiki/parts/5/5a/SecretionofPETase.jpeg
 
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>
  
 
===Sequence and Features===
 
===Sequence and Features===
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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.
 
https://static.igem.org/mediawiki/parts/thumb/6/60/T--Tianjin--partwt.png/800px-T--Tianjin--partwt.png
 
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.