Future Work

Protein Modification

We have gained several mutants of PETase, PET hydrolysis activity of which are relatively improved compared to PETase Wild-type. Among them, it’s worth mentioning that I208V shows an activity increase of two-fold. However, all of our mutants up to now are single-site mutant, which have been merely changed only single one amino acid residue. In our future work, we plan to design double-site or even multiple-site mutants according to our single-site mutants. We hope to combine two or several improved sites together into one protein, like I108V and S207T, to further extend the range of activity improvement. The hydrolysis activity of double-site mutants may not be linear addition of hydrolysis activity of the respective single mutants, but based on rational design rationales, the improvement is promising.

Microbial Consortia

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R-R System

We have constructed the novel inclusion body based reporting system and cell lysis regulation system and introduce the TPA positive feedback regulation system to Saccharomyces cerevisiae. However, we have only tested the CpxR promoter and there are much more than only one promoter that can be induced by inclusion body or misfold protein. For instance, the degP promoter, ibpAB-fsxA promoter, etc. These promoters are also in the iGEM parts database (degP:BBa_K339008, ibpAB-fsxA:BBa_K339011). Which one has the best effect is still unknown yet and more experiments need to be done by later teams or people. We use the RFP as the reporting protein because of it’s easy to detect, even by bare eyes if expression level is high enough. However, unless applying the damaging way like centrifugation or expensive device like 96-well Microplate Reader, the phenomenon is still not so clear. A more intuitive reporting strategy need to be developed. We used the ddpX gene to automatically break the cells, however, it might be better to keep the bacterial alive and make the misfold protein recovered by itself. There have been some researches about this, and some particular enzymes in E.coli can help protein folding and inclusion body degrading in envelope such as degP, fkpA, PpiA, PpiD, DsbA, etc.[1] The TPA positive feedback system is verified by our experiments, but the detailed mechanism has not been clearly found out. We are looking forward to more researches on this field so that we can not only get TPA induced gene, but also genes induced by other substances via sequence design according to the inducing mechanism.

Reference

[1]Tracy L. Raivio, Michael W. Laird, John C. Joly and Thomas J. Silhavy. Tethering of CpxP to the inner membrane prevents spheroplast induction of the Cpx envelope stress response. Molecular Microbiology (2000) 37(5), 1186-1197