TJUSLS-China iGEM team is dedicated to addressing the plastic pollution crisis. Based on previous successes, we are deploying a series of strategies to enhance the production of PETase (variant: LCC-ICCG). We conducted extensive literature research and followed the iGEM engineering cycle (Design, Build, Test, Learn) to form a full workflow of plasmid construction and random mutation through PCR; followed by protein fluorescence quantitation and final high through-put screening and mutant design.
To start with, the pET26b-ICCG (without pelB signal peptide) was chosen to be a reform target and that is constructed the GFP fusion protein. Moreover, we also tested with variety of vectors (pET22b, pET28a, pET28as, pET32a, pMAT9s, pGEX-6p-1, and pCold-TF) and E. coli expression systems (BL21(DE3), BL21(DE3) pLysS, SoluBL21(DE3), Origami2) to enhance the protein yield. Better parameterized signal peptides were obtained by model prediction of the signal peptide database. A high-throughput screen on expression levels following plasmid construction revealed the best nfaA signal peptide and further design optimized by computational modeling and empirical validation. Additionally, we modified the T7 promoter and introduced random mutations into the tac promoter via PCR. The experimental data from wet lab work informed our models, allowing us to refine and complete the engineering cycle.
Our efforts have yielded promising results, and we are now pursuing further exploration toward industrial-scale fermentation. Our ultimate goal is to increase the production of highly active PETase, contributing to the fight against plastic waste. We remain committed to discovering new possibilities for this solution in the future.