Research Projects

Since the discovery of IsPETase ( derived from I. sakaiensis 201-F6) in 2016, there have been several researchers endeavoring to over-expressing the PETase to site-directed mutagenesis for improvising structural and functional stability. The engineering intervention mainly targeted the bacterial host and addressed the following issues: 1)Extracellular secretion 2)Development of stable enzyme 3)Increasing catalytic activity and substrate-specificity However, to maintain the bacteria in the natural environment, the specific carbon sources need to be included on-site, leading to other operational issues. Therefore, recently (since 2019), photosynthetic microbes are gaining attention as hosts for engineering the PETase; in two earlier such efforts ( Kim et al., 2019 and Moong et al., 2020), the PETase was successfully expressed in the photosynthetic microalgae. Also, found active in the organisms growing in seawater containing medium. The present proposal aims to address the lacuna of the above studies -In the first report, the PETase was expressed intracellularly. -The second report addressed the issue leading to the secretion of PETase in the culture medium and hence a reduced catalytic stability compared to the previous report. Our research work aims at the engineering of surface active PETase in organic silaffin matrix. For that, we have chosen T. pseudonana, a marine-centric diatom due to the following two reason -Availability of whole genome sequence data. -Ease in cultivation at environmental temperature. For operational ease, the study utilizes CRISPR based gene editing over the conventional cloning approach. It would be achieved by targeting the delta-5 elongase gene for PETase knock-in. Further, the cas9 target sites with low homology or no homology to other gene loci would be identified using publicly available CRISPR/Cas9 target-finding tools. Next, the sgRNA will be cloned in a diacas9 vector with the fcpA promoter to construct the fcpA-sgRNA expression cassette. Recombinant PETase(silaffin PETase fusion) will be cloned in a pha1 vector for gene insertion to generate the donor plasmid . Next, the engineered vector would be transformed into the Thalassiosira pseudonana, and the transformants are selected directly using Zeocin-resistance in the clone, following gene-specific PCR. The Protein expression and validation would be confirmed by performing SDS-PAGE and Western blot analysis. Further, the catalytic activity and operational parameters would be optimized. The project envisages the clear goal of engineering the PETase on the silaffin matrix of Thalassiosira pseudonana. Upon successful engineering the recombinant T. pseudonana could be used for bioremediation of microplastics in water mesocosm. The expertise of the PI in creating gene fusion would further be helpful in executing the project objectives.