Team:TJUSLS China/Partnership

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PARTNERSHIP

Overview

In the process of moving forward, partners are very important. This year, we have become partners with BJEA_China and DUT_China. We have shared objectives—to completely degrade PET (including areas with high crystallinity and low crystallinity). During 7 months throughout the season, we had many online exchanges, an offline meeting, and completed some experiments together. In the end, we each achieved engineering success, and plan to apply the results of the three parties to the industrial recycling of PETase after the iGEM competition.

With BJEA_China

Click here to visit their wiki

Goal Setting

• At the beginning of the iGEM competition in 2021, we heard that the high school student team of BJEA_China was very interested in PET degradation. At that time, we have already done and research topics related to PET plastic degradation. So we emailed them. After that, we established a WeChat group to discuss our project goals through WeChat. In the process of literature research, we found that commercially available PET has high crystallinity and low crystallinity regions, which can be specifically degraded by PETase and LCC respectively. Therefore, we decided through consultation that BJEA_China will work on the transformation of LCC and TJUSLS_China will work on the transformation of PETase. Our ultimate goal is to use two proteins at the same time to achieve complete degradation of all areas of PET, including areas with high crystallinity and low crystallinity.

Experiment

• After successfully getting in touch, we learned that the BJEA_China needed PET flakes to continue their lab, but what they lacked the most were funding and time. PET films, one of the most necessary experimental materials in their laboratory, is either too expensive or requires an extremely long delivery time. What our team will do this year is to rationally design mutations to improve the stability of PETase. Therefore, we enthusiastically provided them with a sufficient amount of PET film to help them promote the progress of the experiment.
• In addition, before the BJEA_China arrived at Tianjin University, we also helped them complete the trial expression of the protein, laying a solid foundation for the next experiment.
• On July 25, BJEA_China came to our lab to conduct protein purification experiments, which is an extremely important part of the overall experiment.
• On July 26, both teams got up early and went to the laboratory to inoculate the bacteria into the LB solution in the small test tube. At noon, we transferred the LB solution together with the bacteria cultured from 6:00 in the morning, and put it into a large Erlenmeyer flask containing the LB solution. In the afternoon and evening, we systematically explained how to use the autoclave and how to balance correctly for the students of BJEA_China. More importantly, we taught BJEA_China students the centrifugation process and the collection of bacteria, and they completed the protein induction.
• On July 27, the partners of BJEA_China independently did centrifugation and collected bacteria. Later, we taught them the process of protein purification and worked with them on the steps of protein purification. We moved together between the cold storage at 4℃ and the laboratory at room temperature. The process of purifying protein is difficult and long, but working with each other made it a great source of joy and excitement for both teams😀.We truly cared about each other. Worried that our partners would catch a cold, so we borrowed thick coats** from the campus security center. Eventhough everyone was busy and tired, we could still feel the warmth of body and soul in that cold room.
• On July 28, they ran the albumin glue to test the results. They encountered several problems and found that the albumin glue was difficult to decolorize. In the end, we helped them solve the problem and got a satisfa**ctory result.**
• On July 30, TJUSLS_China and BJEA_China held a summary meeting in the School of Life Sciences, Tianjin University. After a week of laboratory experiment cooperation, this summary will allow us to learn more about each team member of our two teams and discuss whether we have other opportunities to help each other to achieve the goal of one plus one greater than two.At the meeting, our two teams systematically introduced our project and showed our PPT to everyone through the Tencent conference. On the one hand, the partners of BJEA_China explained the two methods they used, namely the cell surface display of mLCC in Bacillus subtilis and the joint mLCC with hydrophobins. After that, we used PPT to demonstrate our project for them. We improve the thermostability of PETase by rationally designing mutations to improve the degradation ability of PETase, including the stability of engineered disulfide bonds, hydrogen bonds, salt bridges, and stabilization of the hydrophobic core.After the explanations of the two teams, we gave them some suggestions and support. For the experimental part, through thinking about PETase, we suggest that they choose other hydrophobins or linkers to see if they can get higher efficiency. We also recommend trying to link their LCC with our PETase to check the efficiency and create opportunities for the results of our two projects.
• In addition, during the period from October 1st to October 7th, because BJEA_China was unable to conduct experiments, we helped BJEA_China determine the degradation efficiency of their modified LCC under different concentration gradients, and sincere congratulations on their successful results.

BJEA, we want to say to you:

Although the time for experimenting together is short, through this deep partnership experience, the partnership between our two teams has become deeper. Thanks, BJEA, for the LB solution in many large-size Erlenmeyer flasks you have prepared for us and for the thank-you card you wrote for us. We hope that both of our teams can move forward and achieve success.

Modeling Symposium

• On July 28, 2021, we communicated with BJEA_China in the modeling part to solve the problems they encountered in the modeling part and see if there are new opportunities to help them improve based on the status quo.
• Initially, they shared some ideas about modeling. Since they are the first team to participate in the iGEM competition, they are relatively lacking in modeling experience and knowledge base. Therefore, the members of our modeling team actively communicated with them, helping them to verify their ideas and basic principles on how to make a model predict the degradation rate of enzymes under different conditions. Then, the member from our modeling group also provided them several helpful suggestions. For the available resources, we recommended the CSDN algorithm resource site to them, which provides a reference for their algorithm construction and also allows them to obtain additional algorithm knowledge. More importantly, the members of our modeling team told them a key point. They not only need their experimental data, but also a lot of other data related to their topic. In addition, we also teach them how to search for paper resources, how to search for data, and what to pay attention to.
• During the summary meeting, we discussed the part about modeling. We think we can try to find a suitable hydrophobin through simulation analysis. In addition, knowing that they are immature in computer capabilities, we are happy to provide them with our code for simulation analysis to help them achieve their goals. More importantly, in the final demonstration of their project, we recommend that they use PyMOL to create 3D dynamic graphics to show their project more vividly.
• Finally, we told BJEA_China partners some efficient modeling techniques, and together analyzed some models that won the modeling award.

Cooperation On Human Practice

• After the laboratory cooperation, due to the strong relevance of the research topics, we have a deeper connection. Before the deadline for the promotional video, the two teams decided to send the promotional video, and we exchanged opinions. This move not only shortened the distance between the two teams but also gave us a stronger bond. After sharing the promotional video, we and the BJEA_China team have considered arranging a week to face each other. As a result, the human practice group members of the two teams did not waste any time. We decided to jointly organize a small seminar to share our views on the main concepts of iGEM human practice and event organization strategies.
• On July 26th, our team and BJEA_China both proposed our research topic. Since the research topics are closely related, we have great opportunities for cooperation. From the brief introduction of the two teams, we realized that the two teams researching plastics have the same purpose for protecting the environment. As a result, the HP team of the two teams started the current joint promotion of synthetic biology and education on the importance of plastic handling. Subsequently, we began to organize and distribute the content of the cooperation. At the end of the symposium, we put forward many more feasible ideas, including a documentary documenting the environmental impact of untreated plastics, a teaching course outlining the basics of synthetic biology, and a live podcast to discuss the effects of plastics on a wider audience. The impact of the environment and the necessity of plastic degradation.
• All in all, the similarity of the themes created a stronger connection between the two teams and allowed us to stay in close contact for further cooperation.

Success of Both Teams

• Both teams have established extremely deep partnerships in all branches of iGEM. Laboratory experiments, HP's ideas, modeling work, and other different aspects of work are closely related to BJEA_China through this in-depth partnership.
• In our summing-up meeting, BJEA_China shared with us the results of their successful experiments, which made both of us very excited.
• In addition, at the Plasticase exchange meeting, we listened to each other's project and exchanged the experimental results of the project together. At the same time, TJUSLS_China has also achieved Engineering Success. We express our sincere appreciation and blessings for the achievements of both parties, and look forward to the use of two enzymes to jointly carry out industrial recycling of PETase with various crystallinity in the future.

Friendship lasts forever

• Our team led BJEA_China to visit the beautiful campus of Tianjin University. During the visit, a deeper and more harmonious relationship was established between the members of the two teams. There is no doubt that we not only laid a solid foundation for further win-win success but also gained new friendships.
• Here we want to thank BJEA. Thanks very much for the thank-you card you wrote for us❤. It is an extremely precious gift. Thank you for this incredible journey of iGEM partnership.Looking back on the course of cooperation with BJEA_China, our partnership is very meaningful and unforgettable. We hope that both TJUSLS_China and BJEA_China can gain good results in this year's iGEM, and we believe that the friendship between our two teams will last forever.

With DUT_China

Click here to visit their wiki

Goal Setting

• The DUT_China team learned that we are going to transform PETase this year, and they expressed interest and sent us an email to indicate that they also want to degrade PET. Then, they showed us through online chat that they are going to make a fusion protein of PETase and MHETase. The main product of PETase degradation of PET is MHET, and MHETase can further degrade MHET into TPA and EG small molecules. The DUT_China team is committed to the connection of the two enzymes, PETase and MHETase, and TJUSLS_China will be committed to the transformation of PETase. Therefore, our projects are very close. Our ultimate goal is to fuse the modified PETase and MHETase to achieve the complete degradation of PET.

Partership Throughout the Whole Session

• After DUT_China and TJUSLS_China have conducted many online exchanges, we have established a partnership and reached a consensus with each other. Moreover, our partnership is not limited to the communication of the project process, we also provide more support for each other, and have cooperation in experiments, modeling, HP work, etc., and together contribute to the success of each other's projects.
• In March, DUT_China and TJUSLS_China established a partnership. We learned about each other's projects and set a common goal.
• In April, through online methods such as QQ and email, TJUSLS_China and DUT_China exchanged and cooperated in the direction of degradable PET plastics. We also exchanged some questions about modeling together, and they also introduced us to their teachers so that we can consult.
• In May, we invited them to Tianjin University for offline exchanges and discussions. After DUT_China arrived at Tianjin University's School of Life Sciences, we conducted offline exchanges and discussions about the projects of both parties. Therefore, during the exchange, we got a deep understanding of each other's projects, clarified the joint cooperation points of the two teams' projects, and put forward suggestions for both parties' projects. DUT_China raised questions and improvements in the early stages of our project. We suggest that you can use their skeleton system to replace their PETase with our optimized PETase. After the exchange meeting, we also took the friends of DUT_China to visit the beautiful campus and visit Tianjin together. We had a happy and wonderful time.
• From mid-May to October, our two teams jointly visited offline companies. During the experiment of both parties, they both cared about each other and continued to communicate online to help with the problems of the project. We mailed PET films and the modified PETase plasmid to DUT_China; DUT_China sent us the MHETase plasmid to connect our modified PETase and MHETase. In terms of modeling, we shared with them our suggestions and experience during the web page construction period.
• TJUSLS_China and DUT_China are members of Plasticase, a community dedicated to solving the problem of plastic pollution. At the virtual round table "Reshaping“ the Future, both parties introduced their projects. The other side congratulated us on our success. At the same time, We also send our blessings to DUT_China and hope that they can get good experimental results.