In this year's project, we have strengthened our contact with other iGEM teams and carried out our cooperation activities through the network or offline. In the process of communication, we got a lot of satisfactory results and enjoyed the atmosphere of communication. We hope that through communication and cooperation, we can share our project with other teams and spread the project concept of environmental protection. We hope that more people can understand the harm of plastic waste, and at the same time, we can apply synthetic biology technology to solve the problem. Of course, in the process of cooperation and exchange, we also learned a lot, saw many excellent teams and their creative ideas.
| Cooperation of KEYSTONE  |
In this iGEM project, in the field of cooperation, we realize that the object of cooperation can be further expanded, so that we want to make our work affect more people, so that more people can benefit from it. Therefore, we chose a high school team (KEYSTONE) for project cooperation. In the project, we all chose the theme of plastic biodegradation. There are many things in common. In addition to the experiment, we hope to provide some experience, and we also hope that the previous experience can help the high school team.
At the beginning of iGEM, our two teams had a project exchange. KEYSTONE, a senior high school team, interviewed Professor Liu Luo, the Primary IP of our team, and provided them with some very good project suggestions after understanding their projects.
KEYSTONE’s interview with prof. Luo Liu
At the same time, the keystone team also gave us a lot of help. In the process of making the project promotion video, I would like to thank Jane Liu from the keystone team for providing us with a very good hand-painted video to help us complete the production of the promotion video.
Promotion video drawing for BUCT
At the end of September, we also carried out offline communication activities, in order to further understand the project implementation situation of our two sides and the discussion of relevant experiments. We choose the form of offline communication, which is more conducive to enhance our mutual understanding. We all enjoy the atmosphere of communication and discussion in such a relatively relaxed environment. In the process of communication, first of all, we learned about the progress of our project. During the communication process, KEYSTONE put forward to us the problem that they found that the degradation process of PET was not efficient. According to our knowledge and relevant literature, our team put forward some suggestions to KEYSTONE: pretreatment of pet, optimization of experimental conditions (pH, buffer, T) , to improve the pH and thermal stability of the enzyme, to reform the enzyme catalytic center, and to optimize the experimental methods. At the same time, when we are worried about how to apply our work to real life in the future, KEYSTONE provides us with their interesting hardware equipment ideas, using solar energy as energy supply, integrated design and other good ideas, providing us with new thinking direction for subsequent applications.
In mid October, we were invited by KEYSTONE to participate in the small offline simulation jamboree. We had offline communication with many excellent teams, and introduced our respective projects in the form of posters and slides. We had an in-depth discussion with KEYSTONE again. We shared with them our previous experience in competition and exchanged the production of project videos. At the same time, we also browsed the wiki contents of our two teams. We put forward a lot of suggestions to KEYSTONE in Wiki production. In terms of video production, the artists of KEYSTONE also provided us with their ideas. In the previous video production, we both like the hand drawing form provided by KEYSTONE. In our Team Presentation Video, they also provided us with their interesting hand drawing process again.
In this cooperation, we have established a good partnership. KEYSTONE provides us with an ideal hardware design. They designed a hardware device with solar energy as the main energy supply and can degrade pet. Similarly, our PE degradation can also be realized in this device. With the help of KEYSTONE, we can apply our degradation module and synthesis module in this system. At the same time, we have developed a biosynthesis part, which has been verified by experiments to be feasible. We have constructed a polymerization system composed of CoA ligase and acyltransferase, which can realize the esterification of hydroxyl group and carboxyl group.
The specific reaction mechanism is as follows:
The KEYSTONE project produces mainly MHET after the degradation of PET, while MHET has hydroxyl and carboxyl groups, which can be used for subsequent polymerization with our parts to obtain some new products.

Our two teams hope to work together to achieve biodegradation of various plastics and reproduction of high value-added products by sharing our respective work.
| NJTech_China  |
We are very glad to have a project exchange with team NJTech_China from Nanjing University of technology. This year, we have chosen the mode of exchange and cooperation based on experts' suggestions. We shared our project ideas with each other. After understanding each other's project design, we put forward the possible problems of each project and consulted the relevant experts of the other team.

The Nanjing University of technology team(NJTech_China)asked us their questions:
1. CRISPR/cas9 gRNA design, what are the common gRNA design platforms? What parameters should be paid attention to when selecting? How many gRNAs do you usually use at a time to knock out a gene?

2. Among various gene expression regulation methods, the characteristics and advantages of promoter regulation? What research achievements have been made in promoter engineering?

3. Under normal nutritional conditions, compared with other signal transduction processes, mating specific signaling pathway is relatively independent of the asexual life cycle of yeast. We believe that the pathway can be modified to obtain chassis cells that can respond to specific induction conditions/signals without affecting the growth of yeast, which can be used in biosensors, fermentation condition control and other fields. Do you think the idea has a certain application prospect? In the transformation from current theoretical research to practical application, what problems need to be considered? Can you provide some suggestions and suggestions?

Professor Liu Luo, our team's primary PI, gave the suggestions:

1. CRISPR has some online platforms for gRNA, which can be found by yourself. gRNA will be designed according to PAM sequence, and the ones with better specificity will be selected as far as possible to avoid missing target. If the specificity is good, usually one is enough.

2. This problem is a little too big and contains a lot of content. Promoter is a step of regulation, RNA regulation, translation regulation and so on.

3. The promoters used for biosensors and fermentation condition control are generally of rapid response type. Those related to growth are relatively slow, while those related to metabolism are relatively fast. There are more promoters in S. cerevisiae, so it can be studied from the aspects of response speed, intensity and dynamic range.

At the same time, we also raised our questions:

1. We think that the pathway is too long and related to long-chain fatty acids, so there may be some problems in the solubility of substrates, the ability of enzymes to recognize long-chain and catalytic efficiency, the intracellular hydrophobic environment, and the burden of double plasmids.

2. A large amount of NADPH reducing power is needed, which may be insufficient.

3. The first step is how to make E. coli ingest the decomposition products of laccase? Need to build some transporters? Or does it break down cells for whole cell catalysis?

4. High frequency of T7 promoter may lead to low expression efficiency.

5. The activity of each enzyme in the subsequent polyester synthesis step.

6.Can we insert some genes providing reducing power into the genome of bacteria through CRISPR to improve the reducing power?

Professor Ji Xiaojun, from NJTech_China, gave the suggestions:

1. In view of the problem of long pathway and double plasmid burden, it is suggested to use mixed bacteria fermentation to split the metabolic pathway into different cells to achieve division and cooperation. Attention: control of fermentation conditions in mixed culture system.
For the solubility of long-chain fatty acids, we assume that FA is a metabolic intermediate. We suggest that we should pay attention to the regulation of enzyme content in upstream and downstream reactions, maintain the balance between the production and consumption of long-chain fatty acids, and reduce the accumulation of intermediates.

2. If E.coli is used as the chassis cell, the enzyme that catalyzes NADH to produce NADPH can be overexpressed, or the HMP pathway can be strengthened to improve the synthesis of NADPH.

3. Cell free catalytic system can be used for cell lysis.

4. Firstly, it is suggested to use T7 promoter to express reporter gene, and test the gene expression efficiency under different T7 usage frequency. Secondly, the decrease of T7 expression efficiency may be due to the short supply of trans acting factors required for T7 to play a role, which is related to the excessive cell load, so mixed fermentation can be considered.

① Regulation of enzyme expression → regulation of gene expression (which may be related to our promoter Engineering);
② Modification of the enzyme, such as adding PEST sequence at the C-terminal, can shorten the half-life of the enzyme;
③ In order to reduce the loss of substrate and the accumulation of toxic intermediates, scaffolds can be used to co localize a variety of enzymes in a series of related reactions catalyzed by multiple enzymes;
Concerns: speed limiting steps (key enzymes), control of enzymes that catalyze branching pathways (inhibition of branching pathways).

6. The construction of reductive force synthesis pathway further aggravates the metabolic burden; moreover, the energy metabolism of cells is highly controlled, and the expression of enzymes related to reductive force synthesis is also regulated in many aspects, so the integration of genes may not be able to obtain a large amount of reducing power. In addition, it is suggested that further consideration should be given to the alteration of cellular energy flow which may lead to the disorder of intracellular metabolic flow.

At the same time, our team also got new inspiration from the communication with Nanjing University of technology. Because of the whole metabolic pathway we designed, we need a strong supply of reducing power. Therefore, we are very worried about the insufficient reducing power provided by bacteria. After understanding their projects, we realize that we may be able to insert the genes corresponding to those enzymes that provide reducing power into the genome of bacteria through CRISPR technology, so as to improve the ability of bacteria to provide reducing force. This will be our future research direction.

| TJUSLS_China  |
This year, we are also very lucky to be with tjusls_ China cooperation, Both of our teams are committed to solving the plastic problem, we think our projects are meaningful, and we are willing to use our knowledge to change our world and make us live in a more beautiful world.

We held an online exchange meeting, and we introduced our projects to each other in the form of slides. After the exchange of projects, we all realized that the degradation efficiency of bioplastics was relatively low, which must be solved. From the team of Tianjin University(JUSLS_ China), we have got good suggestions. They suggest that we can improve the thermal stability of the degradation enzyme in the future experimental improvement. The transformation method used in this year's project of JUSLS_ China will provide us with feasible ideas and methods for future experiments. We can improve the thermal stability of key enzymes by modifying disulfide bonds. At the same time, we also discussed the pretreatment methods of plastic materials before degradation. For example, we shared the feasible pretreatment methods: alcohol treatment, alkali treatment and the use of ultrasound to help accelerate the speed of plastic pretreatment.

At the same time, we also introduced our project to them. We innovatively used the surface display technology of Bacillus subtilis. We also believed that the thermal stability of enzyme could be improved by surface display through preliminary research, so we suggested TJUSLS_ China could consider the use of surface display technology in their research, using a variety of methods to improve the thermal stability of petase, but also can play a role in enzyme immobilization. We believe that our experience in building a surface display system can help them in their future research.

Similarly, we also report to TJUSLS_China recommended the part we synthesized, after they had completed their work on the modification of PETase enzymes.We can make use of our part to carry out related work, using the products degraded by PETase as reaction substrates, and using our constructed synthetic part to produce new polymers, to truly realize the recycling of waste.TJUSLS_China is also very happy to use the part we provide in their subsequent work, which will also provide us with more experimental data to further verify the reliability of our part.

We and KEYSTONE_ A had an online communication, and we were very interested in their project BC Bio-patch design. They introduced to us a kind of magic plaster they designed, which takes cell cellulose as the carrier and produces cytokines with specific curative effect through the attached active cells to achieve therapeutic effect. We like their creative design very much. At the same time, we also introduced our project to them, shared some interesting experiences in laboratory work and the stories of participating in iGEM in the past. Of course, we are all aware of the tremendous achievements of 3D printing in the field of biomaterials, and we have come up with 3D printing technology in the projects of both our teams. At the same time, we also face the same problems, such as: how to ensure that the 3D printing process will not be contaminated by bacteria; how to solve the biocompatibility of materials, and so on, which will become the problems we need to solve together in the future.