Team:AHUT-ZJU-China/Engineering
AHUT-ZJU-China
Engineering Success
Research
AHUT-ZJU-China team is committed to the study of CO2 capture. By consulting a large number of literature and analyzing its unique carbonic anhydrase from different organisms , we finally selected OT3-CA-WT as our project after comparative analysis. We found that OT3-CA-WT has good thermal stability and can maintain good catalytic activity even at higher temperatures, but its catalytic activity is very different from that of human beings.
Fig. 1 Three-dimensional structure of OT3-CA-WT protein
Design
Therefore, we decided to use the method of synthetic biology to mutate OT3-CA-WT so that it can improve its catalytic activity on the basis of its original thermal stability.
Fig. 2 OT3-CA-WT’s active site
Construction
We used Escherichia coli as engineering bacteria, constructed the vector with pET28 a(+), and successfully expressed OT3-CA-WT-His with high efficiency. At the same time, we also constructed the mutant vector and predicted its expression effect.
Test
We used PyMOL, ADT and other software, and analyzed the original OT3-CA-WT amino acid sequence and three-dimensional structure. On this basis, we predicted the mutation site, and successfully simulated the change of its catalytic activity after a series of tests.
Study
All of us have learned a lot through this project. First of all, we learned how to use some unique biological software. These software can help us to make reasonable inference and prediction before the formal experiment, reduce meaningless attempts, and save the time and cost of the experiment. At the same time, through the experiment, our understanding of synthetic biology has risen from theory to practice, and with the combination of theory and practice, we have deepened our understanding of synthetic biology.
Throughout the course of the project, all members are no longer confused about the concept of Greenhouse Effect. We realized that besides using the traditional physical and chemical methods to solve problems, it has advantages to use synthetic biology to transform organisms to achieve our goals. In the near future, we human beings will give full play to our imagination and use synthetic biology to solve more thorny problems.
Improvement
We mutated six amino acids of OT3-CA-WT on the basis of the original: 122: P→K (the 122nd proline is mutated to lysine), position 123: P→K (the 123rd proline is mutated to Lysine), position 128: P→K (mutation of the 128th proline to lysine), position 132: L→K (mutation of 132nd leucine to lysine), position 134: L→K ( Leucine at position 134 was mutated to lysine), and position 137: P→K (leucine at position 137 was mutated to lysine).
After software simulation and prediction, we found that the OT3-CA-MU activity of the mutation was improved, which was consistent with our expected goal.
Fig. 3 Structure of OT3-CA-WT and OT3-CA-MU
Research
Looking at the overall situation and through summary and reflection and re-review of the data, our project has been successful in terms of simulation and prediction this year. Due to the COVID-19 's pandemic situation, our laboratory was not open before, we cooperated with Zhejiang University in our experiment project.We also carried out a large number of simulated mutations on the computer using software such as PyMOL, ADT, analyzing the amino acid sequence and structure of the OT3-CA-WT, we predicted the simulated mutation sites, and finally got our results after a series of tests. The overall energy of the OT3-CA-MU was higher than that of the previous wild type, and the original binding site and Zn2+ catalytic site were not destroyed and could still achieve the original function. Therefore, our simulation prediction is successful from an engineering point of view. We have high hopes for the future. Even if the competition is over, we will continue to experiment and improve the simulation results. We firmly believe that eventually it can become a product that can really capture a large number of CO2, benefitting the mankind and the whole planet.