Nowadays, plastics are widely used around the world, and polyethylene terephthalate (PET) has caused serious pollution to the environment. To degrade PET materials without causing other kinds of pollution, we try to set up a biological degradation platform by using PETase. So that the yield of protein we obtained is really important. In order to achieve improving the yield of PETase, we searched the iGEM Biological Parts library for secretion peptides and picked BBa_K2013021, pelB. This is a biological part submitted by iGEM2006_Duke in 2006, and they chose pelB to transport protein to the extracellular culture medium. However, the yield of the target proteins is not enough for use.
Our team increased another peptide to complete the secretion system and test its function. This information can be a good reference for future iGEM teams working on protein secretion in the E. coli system.

PelB (pectate lyase B signal peptide) is a Sec-dependent translocation signal peptide, it contains 22 amino acids, and it is commonly used for the production of extracellular recombinant proteins in E. coli. In our project, we fused pelB with SbPETase to achieve SbPETase secretion.

In order to increase the yield of PETase in the extracellular culture medium, we constructed two recombinant plasmids: 1. fused pelB at the N-terminal of SbPETase and ligated it to the double-enzyme digestion pET22b vector, 2. Insert Kil gene into the pRSFduet1 vector. After identifying the correct colony through enzyme digestion, we sent the corresponding plasmid to the company for Sanger sequencing.

To test if Kil signal peptide can improve the yield of SbPETase proteins in the culture medium, we also co-transformed the recombinant plasmids pET22b-pelB-SbPETase and pRSFDeut1-Kil into E. coli BL21(DE3), expanded the culture in the LB medium and added IPTG to induce protein expression when the OD600 reached 0.4. After overnight induction and culture, we purified the protein SbPETase we wanted and collected the data (Figure 2). In Figure 2, when co_transformed pET22b-pelB-SbPETase and pRSFDeut1-Kil, the yield of protein SbPETase in the extracellular medium is higher than without it.

SbPETase is a novel enzyme for Poly (ethylene terephthalate) (PET) hydrolysis. This hydrolase can degrade PET into small fragments, then transport the degraded products for further "digestion", and finally convert them into two relatively simple organic compounds, ethylene glycol, and terephthalic acid.

SbPETase is an efficient enzyme for PET and BHET material, and SbPETaseW132H is developed when we did the site-direct mutation of SbPETase. When detected the activity of degradation of PET, SbPETaes_W132H showed more activity and can be a candidate for future PET degradation research.

pRSFDeut-vector is one of the most commonly used E. coli protein expression vectors, which uses the T7 promoter to regulate the expression of exogenous genes. The vector is a high-copy-number plasmid. When expressed in the prokaryotic system, the Kana+ resistance can be used to screen the right colony and can insert target genes in MCS. This plasmid backbone can be used to express different proteins in the future.

Our team has carried out a series of preparations for plasmid extraction, preparation of receptive state, and purification of protein. Then we implemented PCR amplification and then verified the target gene fragment by agarose gel electrophoresis, and finally recovered the plasmid. After that, the vector was digested by double enzymes, verified by agarose gel electrophoresis, and the plasmid was recovered. The team assembled and transformed the recombinant plasmid and identified it after culturing it overnight. The construction of the SbPETase mutant plasmid was done by the team after. When the construction was completed, we continued to verify. Then we transformed the recombinant plasmids into E. coli BL21 (DE3), purified the protein and detected the activity of SbPETaes in vitro. Overall, our study provides a foundation for accelerating the discovery of novel PETase variants screening platforms for industrial applications.