Part:BBa_M50056:Design

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 [1]. Thus, we decide to use the 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 promoter 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 [2]. The two mutations designed by the Tianjin team are the 208th amino acid (changed from isoleucine(I) to valine(V)) and the 90th amino acid (changed from arginine (R) to alanine (A)) [2]. 6 His tag is also appended at the end of the PETase sequence for western blotting test. Wild type PETase from Ideonella sakaiensis is identified by Yoshida et al. [3] and the mutated PETase gene here is optimized for E.coli with IDT’s codon optimization tool. The purpose of our codon optimization is to imrpove the expression of PETase gene since it is not originally from E.coli.

Source

Wild-type PETase sequence is from Ideonella sakaiensis and is identified by Yoshida et al. [2]. Two mutation sites are originally designed by 2016 iGEM Tianjin team [3]. Most of the other parts of this composite part, rhamnose-inducible promoter, an ampicillin resistance marker, an origin of replication, a secretion tag, and a strong RBS, come from DNA 2.0 [4].

References

[1] IGEM16_Harvard_BioDesign. "Team:Harvard BioDesign/Basic Part." Team:Harvard BioDesign/Basic Part. 2016 Harvard IGEM, n.d. Web. 12 Dec. 2016.

[2] Yoshida, Shosuke, et al. 2016. "A bacterium that degrades and assimilates poly (ethylene terephthalate)." Science 351.6278: 1196-1199.

[3] IGEM16_Tianjin. "Part:BBa_K2110105." Part:BBa K2110105. IGEM, 12 Oct. 2016. Web. 11 Dec. 2016.

[4] "Bacterial Expression Vectors." ATUM. ATUM, n.d. Web. 12 Dec. 2016.