Difference between revisions of "Part:BBa K2110800"
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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. | 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. | ||
<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> | <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=== | ||
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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=== | ||
| + | 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
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 79
Illegal BglII site found at 289 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
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.