1. Experimental part

1. XMU CHINA

We met Xiamen University at the CCIC Conference on Synthetic Biology in China.

In this year's project design, we involved the blrA and pBLind-EL222 systems. After reviewing the competition materials of previous years and the iGEM official website, we found that XMU-CHINA had used these two systems in the 2021 season, so we contacted the Xiamen University team for further discussion and exchange.

As XMU-CHINA had chosen to use the toxin blrA and pBLind-EL222 system for the 2021 season in our project design, we reached out to XMU for help in obtaining these genes. In August, XMU presented us with the blrA_pCC-1 plasmid. In addition, we discussed and exchanged ideas about pBLind-EL222, a blue light induction system, and XMU seniors of season 2021 provided us with advice and assistance in experimental design.

EL222 is a light-activated transcription factor that homodimerises and binds to the pBLind promoter under blue light irradiation to initiate transcription of downstream genes. After communication and discussion with the HainanU we redesigned the experimental protocol and based on the lethal gene provided by XMU_China, we designed a plasmid for simultaneous blue and red light irradiation lethality. However, due to the epidemic, HainanU will assist us in conducting experiments on the lethal blrA protein by irradiating the blue light variant alone. In addition, we also held a seminar to share our experience on the experiments and the operational details that we need to pay attention to in order to help both parties to better advance the topic.

2. Hainan U CHINA

In May this year, we found a like-minded partner - the iGEM team of Hainan University. Hainan University this year designed PNA and single base mutation target sequences for base complementary pairing, improved the accuracy of target sequence detection, and enhanced the specificity of the detection system to achieve the ability of single base recognition and detection, and then combined with cas13, cas14, and csm6 proteins, it was used to detect antibiotic resistance genes in bacteria on Hainan Island. On the test of lethal system, our teams have reached a consensus, mainly through the PCR technology used by Hainan University in this project to amplify the target gene, and then carry out the transformation of competent cells, the expansion of transformed bacteria, and protein gel electrolysis test and other experimental methods.

Our cooperation with the experimental part of Haida began on August 1st. During the half-month period from August 1st to August 15th, we had detailed discussions on the experimental content every two days. The expression conditions, culture methods and experimental steps of toxic proteins were all reflected in the meeting. Hainan University will use PCR to amplify the toxic protein gene, and verify the toxic protein expression through transformation, expansion, protein electrolysis and other processes.

In mid-August, we sent plasmoids with toxic protein genes to Hainan University. Hainan-U students successfully received it on August 31, and the experiment started.

Subsequently, we mailed the pro plasmide to Haida students in September. On September 8, Hainan University successfully received the red light-sensitive plasmid and performed the relevant expression.

After conducting several experiments, none of them reached the expected results. After communicating with the Hainan University team, an analysis of the causes was conducted:

1. Not completely avoid light, toxic genes under natural light.
2. Transformation failure due to the inaccurate control of the heating temperature and heating length of time.
3. The number of plasmid bases is too large, self-emesis occurs, and it is necessary to change the sensation.
4. Due to the reverse PCR, the pro plasmid is too long and affected by various factors of dragging and mixing.
5. Enzymes are uncommon.

After fully discussing the experiment and considering various factors, both sides preconceived the subsequent experimental steps: the Jilin University team was responsible for verifying that the blue light promoter could start normally, and the Hainan University team was responsible for verifying that the toxic genes could be expressed normally. In the condition that the blue light promoter can be expressed normally, and the toxic gene can also be expressed normally, and the blue light promoter can be expressed normally, there is the possibility that the toxic gene can be normally expressed.

The control experiment was designed to obtain the normal expression, while the plate long bacteria coated with the bacteria was not transferred to the virulent gene, in contrast to proving the normal expression of the virulent genes, causing the death of the bacteria.

3. BUCT

In the 2022 season, we plan to use E.coli to synthesize 2-phenylethanol. In the preliminary research, we learned that BUCT is also involved in the synthesis of 2-phenylethanol this year, so we contacted the them.

We found in reviewing the literature that the complete route for preparing phenylethanol could shorten the entire plasmide by adding intermediates. So we decided to introduce only the kdka gene and adh1 for the purpose of shortening the plasmide.

JLU_China plan to form an experimental control group with BUCT. In addition to the KdcA gene and Adh1 genes, the Beijing University of Chemical Technology will also introduce a full set of phenylethanol synthesis paths. After the completion of the experiment, the two teams will compare the two results to explore the difference and yield of phenylethanol synthesis between the two methods.

4. FAFU

FAFU involved El222 sequence gene in the selection and preparation of this year's project. At their request, we provided the EL222 sequence-containing bacterial broth to Fujian Agriculture and Forestry.

FAFU was engaged in the topic selection of incense production last year, and their team helped us solve the problem of the death of aromatic alcohols to bacteria and the problem of linalool synthesis.

FAFU's project is the synthesis of phenol ols . The project outline is as above. We communicate with them in online meetings, communicate about the progress of the project, and explore cooperation.

We had a deep and friendly exchange with the Fujian agriculture and forestry team from July to August 2022.

In the experiment, both our team and the Fujian agriculture and forestry team used the blue light-induced suicide gene, and we provided guidance for their gene synthesis and sent the transformed bacteria. The Fujian agriculture and forestry team found some shortcomings in our experiment, such as linalool, which may kill E. coli, and gave very valuable improvement suggestions based on the experimental experience of last year.

We also helped Fujian agriculture and forestry to perfect the lethal gene part in the experiments.

2. Handbook section

HainanU_CHINA

In the early days of the team, we established a good partnership with Hainan University and carried out a long-term communication plan.

As two igem newcomers, we had a lot of problems with new teams in the early days. We think that many problems are in common, so we recorded many basic problems encountered in the process of team formation and competition, and gave our solutions, and compiled this experience manual, to provide reference and help for the later participating teams.

Up to now, our two teams have jointly pushed the content of four public accounts together. With the advance of the tweets and the deepening of the content, we have gradually integrated and completed the version 1.0 manual. In the future, I hope to find more teams to join in on this basis, to share, and constantly improve and expand this experience manual, published in the official community in the form of open source, as a truly help the future igem team to learn, promote the development of igem competition and even the development of synthetic biology manual. In this sense, it is not only a summary of experience, but also a platform for all our teams to exchange experience. At the same time, it connects the past and the future, paving the way and waiting for communication between the latecomers and the pioneers.

At the same time, we also joined the University of Electronics Science and Technology in the later whitelist expansion, which also played an important role in the subsequent manual editing.

3. Public account publicity part

HainanU_CHINA

Together with Hainan University, we regularly release the recent project progress and synthetic biology topics on the official account. These include topics like how to find teachers, how to form a team, how to start it, and how to unite with other teams.

4. The meeting part

1. Lecture section

1.1 Ten schools jointly preached

We held a joint public education lecture with teams from ten universities. After careful analysis, we decided on the topic to be close to the Biology Olympiad and introduce synthetic biology from the competition. This is convenient for many middle school students to adapt to the topic. Then we contacted Beidou Xueyou, a competition tutoring agency, to better expand its influence. At the same time, the ten universities have also established good friends and learned and talked with each other about projects. Our presentation videos were recorded and posted on Bilibili's synthetic biology account from scratch for free viewing indefinitely by all.

1.2 Twelve schools jointly preached

The teams of BNUZH-China, JLU-China, BIT, BUCT, BUCT, NJU-China, FaFu-China, B U C T-China, iBowu, NJMU-China, NPU-CHINA, Sustech_Shenzhen_HCL, and BIT-China fully discussed the course and defined the theme to interest the non-professionals without feeling too rough and simple. In 16 preach, together with many high school and non-synthetic biology professionals, introduces the relationship between synthetic biology and life, synthetic biology hair loss treatment, gene editing and production, synthetic biology and medical care, synthetic biology and energy utilization topics, received unanimous high praise, and helped more students to ignite the enthusiasm for synthetic biology.

2. Modeling section

2.1 LZU-HS-PRO-A

Depression is one of the most common mental illnesses, with over 30 million patients worldwide. It causes hopelessness, or even suicidal thoughts. To help relieve depression, our team developed a probiotic to regulate depression, based on the gut-brain axis. Since it has been previously proven that 5-HTP can increase the concentration of serotine in the brain to improve mood, and GABA can block certain nerve cells to inhibit anxiety and stress, we overexpressed the TPH1 gene into E.coli Nissle 1917(EcN) to catalyze the production of tryptophan which consequently catalyzes the biopterin-dependent monooxygenation of tryptophan to 5-HTP. Meanwhile, we overexpressed the GadB gene in EcN to convert the glutamate to GABA using pyridoxal phosphate (the active form of vitamin B6) as a cofactor. To prevent genetically modified contamination, our team developed a suicide system for the strain. This probiotic has the potential to establish a more peaceful society with higher productivity.

2.2 HainanU CHINA

We also have an experimental modeling section in collaboration with Hainan University. After in-depth exchanges with Hainan University, we intend to jointly establish a mathematical model of DNA double-stranded structure to undo with increasing temperature. At the same time, we helped Hainan University analyze the physicochemical properties of commonly used proteins, and added some models of colony growth fitting and enzyme activity. In the project content of both teams, there are applications to this part of the content.

3. Online Meeting

3.1 DKU_CHINA

We spoke with Duke Kunshan University on our understanding of synthetic biology, and we further hope to organize a Lanting gathering to find more people to exchange projects, evaluate the social value of the projects, and expand the scope of cooperation. Therefore, we met together in early July 2022 to make a preliminary formulation of the format and what needs to be considered. The specific process of hosting the meeting was finalized in August. The Meeting be held in early September.

3.2 CCIC

We participated in the 9th China iGEMer Exchange Conference, at which we introduced the project and communicated and commented with other teams. At this meeting, all of us promoted our upcoming iGEM experience manual, expanded the influence of the team and manual and got a lot of many new teams.