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Overview

This year we focus on improving an essential part, PETase, and has enhance its thermostability greatly(up to 25°C). We totally submitted up to 90 parts of PETase accordingly. After testing with our engineering expression system, we finally confirmed 7 of them that showed satisfactory results. They are Super1(BBa_K3715001), Super2（BBa_K3715002)，Super3（BBa_K3715003)，Super4(BBa_K3715002), Super5(BBa_K3715005), Super6(BBa_K3715004), Super7(BBa_K3715007).

1^st Circuit constructon

Abstract: We constructed 26 pET21a-PETaseMUT(or PETaseSUPER)-His plasmids (BBa_K3715035~BBa_K3715057，BBa_K3715008 ~ BBa_K3715014) , all expressing in E.coli.

Firstly, we want to construct the genes of these 27 mutants. These mutants contain more than ten mutation sites. We felt at a loss when we first wanted to construct the PETaseMUT genes, but we finally succeeded in constructing them through the Overlap PCR method by searching the literature. Here are parts of our results.

Figure1. Results of gene construction

Secondly, we linearized the pET21a, backbone and gene segment to do the homologous recombination, and do enzyme digestion to verify the success of construction. The agarose gel electrophoresis of pET21a-PETaseMUT-His plasmids are shown in Fig.2b, c.

Figure 2 a. Construction of pET21a-PETase-His b.The verification results by enzyme digestion of pET21a-PETaseMUT-His plasmids c.The enzyme digestion results of pET21a-PETaseSUPER-His plasmids

1^st Protein expression

Abstract: Subsequently, the mutant proteins are expressed in engineering bacteria. Super1 to Super7 were purified successfully and could go to the testing step. However, MUT1 to MUT19 forming inclusion bodies, which are hard to use as enzymes.

After Pre-expression and Massive expression, We used the Ni Agarose to purify the target protein. Unlike the expression level of Super1~7, the expression level of PETaseMUT on the vector (pET21a) is very low，according to the result of SDS-PAGE (Fig.4) .

Figure 3. Results of successful expression(Super1 to super7)

Figure 4. Results of inclusion bodies

Gel filtration chromatography: The collected protein samples (Super 1~7) are further purified by Gel filtration chromatography.We determine the state of protein aggregation based on the peak position and collect protein samples based on the results of running the gel.

Figure 5. Results of final purification by Gel filtration chromatography (Super 1~7)

2^nd Circuit constructon

---add tags to help disslove

Abstract: We added different tags in the genetic circuits, constructed 38 circuits with tags of MBP and SUMO. We constructed 19 pMAT9s-PETaseMUT(or PETaseSUPER)-MBP plasmids (BBa_K3715015 ~ BBa_K3715033) and 19 pET28b-PETaseMUT(or PETaseSUPER)-SUMO plasmids, (BBa_K3715059 ~ BBa_K3715077) plasmids, all expressing in E.coli.

Firstly, we want to construct the genes of these 27 mutants through the Overlap PCR method. Here are parts of our results.

Figure 6. Results of gene construction

Secondly, we linearized the pMAT9s and the pET28b backbone and gene segment to do the homologous recombination, and do enzyme digestion to verify the success of construction. The agarose gel electrophoresis of pMAT9s-PETaseMUT-MBP plasmids are shown in Fig.8b, c. The agarose gel electrophoresis of pET28b-PETaseMUT-SUMO plasmids are shown in Fig.7b,c.

Figure 7 a. Construction of pET28b-PETaseMUT-SUMO b.The enzyme digestion results of pET28b-PETaseMUT-SUMO plasmids c.The enzyme digestion results of pET28b-PETaseMUT-SUMO plasmids

Figure 8 a. Construction of pMAT9s-PETaseMUT-MBP b.The enzyme digestion results of pMAT9s-PETaseMUT-MBP plasmids c.The enzyme digestion results of pMAT9s-PETaseMUT-MBP plasmids

2^nd Protein expression

Abstract: MUT1 to MUT19 forming inclusion bodies in the 1st Protein expression. Therefore, we change the tag to SUMO and MBP and optimize the expression conditions to obtain high-expression protein. So far, 26 mutant proteins have been produced and purified as expected.

Optimize the expression conditions: We optimized the protein expression conditions of , involving the concentration of IPTG, and the inducing time as well as temperature. We have tested more than 40 kinds of conditions in total.

Figure 9. Expression conditions

Figure 10. Results of expression conditions’ optimization

After Pre-expression and Massive expression, We used the Ni Agarose to purify the target protein. Unlike the expression level of Super1~7, the expression level of PETaseMUT on the vector (pET28b-PETaseMUT-SUMO ) is still low，according to the result of SDS-PAGE (Fig.11) . PETaseMUT on the vector (pMAT9s-PETaseMUT-MBP) have been produced and purified as expected(Fig.12).

Figure 11. The SDS-PAGE result of PETaseMUT-SUMO

Figure 12. The SDS-PAGE result of PETaseMUT-MBP

Gel filtration chromatography: The collected protein samples (MUT 1~19) are further purified by Gel filtration chromatography.We determine the state of protein aggregation based on the peak position and collect protein samples based on the results of running the gel.

Figure 13. Results of final purification by Gel filtration chromatography (MUT 1~19)

Enzyme thermostability determination

Abstract: We use HPLC equipment to measure the peak area of the product of PET (MHET) of the reaction, in order to express the thermostability of PETase. In conclusion, the thermostability of Super1 to Super 7 has greatly improved compared with WT(wild type PETase). Among them, Super 5 shows the best performance, the released product of which was increased by 163 times. The tolerance temperature of Super5 is increased from 40℃（wild type) to 65 ℃ .

To test the thermostability of our Improved Parts, first, we need to set up a reaction system as shown in the Fig.14. All enzyme reactions were performed in duplicate in 1.5 mL microcentrifuge tubes with a piece of PET Flack, 4.2mg enzyme and reaction buffer at the Optimum pH of PETase, and then were heated from 40°C to 80°C to test it’s thermostability. We used HPLC to analyze the reaction mixture and got the HPLC chromatogram of products as shown in Fig.14. We got its Peak Area by Integration, and then figured out its concentration. Using this method, we tested our mutants.

We heat up from 40 degree centigrade to 80 degree to verify the thermal stability of mutants. We surprisingly finds that Super5 has extraordinary performance at 60 degree taht is 163 times Higher than WT and is still stable at 65°C(Fig. 16~22).

Figure 14. Method we used to test our mutants.

Figure 15. Results of enzyme thermostability determination by HPLC.(MUT1~19)

Figure 16. Results of enzyme thermostability determination by HPLC compared with wild type.(Super1)

Figure 17. Results of enzyme thermostability determination by HPLC compared with wild type.(Super2)

Figure 18. Results of enzyme thermostability determination by HPLC compared with wild type.(Super3)

Figure 19. Results of enzyme thermostability determination by HPLC compared with wild type.(Super4)

Figure 20. Results of enzyme thermostability determination by HPLC compared with wild type.(Super5)

Figure 21. Results of enzyme thermostability determination by HPLC compared with wild type.(Super6)

Figure 22. Results of enzyme thermostability determination by HPLC compared with wild type.(Super7)

Conclusion

This year we have successfully constructed 26 pET21a-PETaseMUT(or PETaseSUPER)-His plasmids , 19 pMAT9s-PETaseMUT(or PETaseSUPER)-MBP plasmids and 19 pET28b-PETaseMUT(or PETaseSUPER)-SUMO plasmids, all expressing in E.coli. Among them, Super1 to Super7 were purified successfully with His-tag and Mut1~19 were also purified successfully with MBP-tag through the effortd of changing the tag and optimizing the expression conditions. Among them, we successfully screened seven PETase mutants（Super1~7) , which greatly improved their thermal stability. One of the optimal mutants (Super5) had an 163-fold increase in enzyme performance compared to the wild-type PETase at 60 degrees Celsius.

Future plan

Our desires to find the better MT would never be satisfied. We are looking forward to gaining even better results after iGEM. Follow-up research in the future will build on these mutants that we have screened(Super1~7) to build mutants that are more thermally stable.