Difference between revisions of "Part:BBa M50056:Design"
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With respect to design on PETase, our design thinking is based on the hypothesis that the combination of the two mutations will further enhance the PETase catalytic activity, since each individual mutant showed improvements of catalytic activity. 6 His tag is also appended at the end of the PETase sequence for western blotting test. The PETase gene is optimized for E.coli with IDT’s codon optimization tool . | With respect to design on PETase, our design thinking is based on the hypothesis that the combination of the two mutations will further enhance the PETase catalytic activity, since each individual mutant showed improvements of catalytic activity. 6 His tag is also appended at the end of the PETase sequence for western blotting test. The PETase gene is optimized for E.coli with IDT’s codon optimization tool . | ||
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Revision as of 05:38, 12 December 2016
We would like to test the catalytic activity of the combination of the two mutations with unlysed cells. Thus, we need to design a secretion tag. The ompT tag secreted proteins into the extracellular matrix, while YebF exported protein into the growth medium. However, YebF was documented to interfere with the PETase activity by the 2016 iGEM Harvard team. Thus, we decide to use ompT tag that is also naturally encoded by E.coli.
We use E.coli as our chassis organism as it grows fast and is one of the most widely studied and used organism for synthetic biology. Our plasmid is synthesized by DNA2.0, and based on our choice of ompT secretion tag and ampicillin resistance marker, the only combination left includes a rhamnose-inducible promotor and a strong RBS, and low E.coli copy.
With respect to design on PETase, our design thinking is based on the hypothesis that the combination of the two mutations will further enhance the PETase catalytic activity, since each individual mutant showed improvements of catalytic activity. 6 His tag is also appended at the end of the PETase sequence for western blotting test. The PETase gene is optimized for E.coli with IDT’s codon optimization tool .
/Users/maihuanghao/Desktop/12_06_single_unlysed.jpg