Difference between revisions of "Part:BBa K5236012"

Latest revision as of 13:21, 2 October 2024

BhrPETase M57L

Plastic pollution poses a serious threat to the global environment. One of the potential solutions, enzyme degradation, would be a suitable approach of dealing with plastic wastes. Among all plastic pollutions, more than 10% of them are Polyethylene terephthalate (PET). Thus, our team has been searching for possible PET hydrolases to break down PET. However, according to Nature's publishment on April 27, 2022, traditional PET hydrolases' enzymatic ability of degrading PET are easily affected by the fluctuation of temperature and pH value. Therefore, we decided to artificially mutate wild-type BhrPETase to increase the enzyme’s range of tolerance so that it can efficiently degrade PET under a wider range of environmental conditions, thereby enhance its potential application. BhrPETase was identified by the Shingo group in a metagenomic study on uncultured thermophiles and was deposited into the NCBI database by the group in 2018 and annotated as a PET hydrolase. As one of the most-confident mutants created in our lab, this basic part encodes mutated BhrPETase M57L.

Usage and Biology

To generate mutated variants, we have trained a Transformer AI model. This model predicts the top 10 potential mutation sites, which are likely to have significant impacts on the enzyme's structure and function. Next, we analyzed the top 10 potential sites via Meta's ESM-1b model to eliminate the silent mutations, which involve changes in nucleotides that do not altering the corresponding amino acids. This ensures that the mutations result in changes in the enzyme's structure and thereby its function. For further information, please check the model page on our wiki. https://2024.igem.wiki/basis-china/model

The BhrPETase M57L sequence is expressed in E.coli BL21(DE3) using the pET28a vector. The pET-28a is a classical plasmid vector used for protein expression in E.coli. This vector contains the T7 promoter, the lac operator, a ribosome binding site, the 6xHis sequence, and the T7 terminator. The T7 promoter is a strong promoter recognizable by T7 RNA polymerase, used to regulate gene expression of recombinant proteins. The lac operator can be activated by IPTG and used to control gene expression. The 6xHis sequence encodes for a tag that facilitates protein purification. Asides from the features included in the plasmid backbone, we added a signal peptide sequence — pELB — before the BhrPETase M57L sequence, which is inserted between the promoter and terminator.

We tested for successful plasmid construction and transformation into E.coli through colony PCR and gel electrophoresis. The following gel result demonstrates that the plasmid transformed into E.coli are correct. The plasmid should have a total of 891 base pairs and the results match.

There were some problems with the sequencing results and the plasmid is being reconstructed.

To test the potential PET degradation efficiency of the BhrPETase N191S synthesized in E.coli we applied the p-nitrophenyl butryte degradation assay from the iGEM19_Toronto team (for more details, please see protocols). The following graph shows the enzyme activities of BhrPETase WT and IsPETase WT compared to the mutations N191S, W229F, N205G.

Reference

Lu, Hongyuan, et al. “Machine Learning-Aided Engineering of Hydrolases for Pet Depolymerization.” Nature News, Nature Publishing Group, 27 Apr. 2022, www.nature.com/articles/s41586-022-04599-z. Kato, Shingo, et al. “Long-Term Cultivation and Metagenomics Reveal Ecophysiology of Previously Uncultivated Thermophiles Involved in Biogeochemical Nitrogen Cycle.” Microbes and Environments, vol. 33, no. 1, Jan. 2018, pp. 107–10. https://doi.org/10.1264/jsme2.me17165

Sequence and Features