Partnership

1.Reach Cooperation Intention

In mid April, our team received an email from NJTech_China of Nanjing University of Technology https://2022.igem.wiki/njtech-china/partnership. The team said that they learned that both teams' projects this year involved strain transformation. Since both teams are in Nanjing, they hoped to cooperate in experiments and hpHP. They advocated mutual learning and win-win cooperation. After several short email exchangescommunications, the two teams roughly knew their respective project contents and experimental directions. The cooperation intention has been reached. It was agreed to hold an online meeting at Tencent on May 25.

Fig1:Content of incoming and outgoing mails

2.Initial Communication & Discussion on Cooperation Plan

At 7:00 p.m. on May 25, the two teams held an online meeting through Tencent Conference APP. This exchange communication was mainly divided into three parts. First, the project was introduced. Then the two teams introduced the progress and problems of the experimental group and HP. In HP, both teams introduced their arrangements and plans. Due to the repeated outbreaks in Nanjing, the two teams said that the follow-up activities would be conducted online and offline. In terms of experiment: We have a general understanding of the research direction of NJTech_China before this line, namely the aerobic transformation of C. butyrate. They want to improve the soil in the anaerobic environment on the planet, prepare for the subsequent arrival of humans and plants, and create the era of interstellar farmers. After learning about their research direction, we conducted a series of related materials to provide them with a possible experimental idea in this online conference: PerR is a hydrogen peroxide sensor, widely distributed in Gram-positive bacteria, belonging to the Fur protein family of metal-binding transcription factors. By combining with the conserved sequence (TTATAATNATTATAA) on the promoter of its regulated genes to inhibitory the specific enzyme activity, the cells fail to initiate their own oxidative stress defense system to scavenge oxygen or reactive oxygen species and fail to survive in the aerobic environment. Therefore, we propose to engineer PerR transcriptional regulators to achieve the aerobic growth of C. tyrolate. The online meeting deepened the understanding of the two teams on each other's projects. Both siThe online meeting deepened the understanding of the two teams on each other's projects. Both sides put forward optimization suggestions on the current progress, and also provided ideas for carrying out more follow-up activities.

Fig2:Screenshot of online meeting

3.Exchange PV Ideas and Provide Suggestions

At 8:00 p.m. on June 25, the two teams held an online meeting through Tencent Conference APP. At this meeting, HP team members shared their ideas on PV and technical issues. . In terms of experiment: we provide the engineered PerR transcriptional regulators to achieve Clostridium butyrate aerobic growth, which was discussed in the last communication. We made feedback after specific experiments: ΔPerR shorten the delay of cell growth under aerobic conditions (100 rpm), and improve its tolerance to oxygen. However, under anaerobic conditions, the growth performance of ΔPerR strain is weaker than the control strain. That's why they abandoned the experimental idea. NJTech_China has learned that we are currently screening different sources of fat lipid elongase / desaturase. C16:0 is about 15% of the TFA of strain Po1f-2. We screened C16/18 elongase gene from two different sources: C16 / 18 elongase gene from Parietichytrium sp. and C16/18 elongase gene from the rat. They are responsible for the elongation of the carbon chain to convert C16:0 to C18:0. Among them, rElo2 has the best catalytic effect on C16:0. However, we wanted to further screen for enzymes from different sources, so NJTech_China recommended the C16/18 elongase gene from Misgurnus anguillicaudatus for us in this online communication. We subsequently applied it to the experiments to look at its catalytic effect on C16:0. As illustrated in following figure. After the exchange, NJTech_China invited us to participate in the opening ceremony of their summer camp.

Fig3:Screenshot of online meeting

4.A Profound Discussion on Experimental Part & Prepare for Joint Activities

On July 26, NNU-China 2022 was invited to attend the opening ceremony of the summer camp, which was organized by NJTech_China. Two sides held an offline exchange meeting then, on the biomedical public technology service platform in Jiangbei New Area, Nanjing. In the exchange meeting, the experiment, HP and education sections were deeply communicated and discussed. After attending the opening ceremony, the two teams exchanged project contents in the conference room. Later, members of NNU-China 2022 visited the laboratory, led by Li Mingxuan, vice leader of NJTech_China. The two sides exchanged in depth the problems encountered in their respective experiments so far. They also said that they could help each other to solve problems. Finally, the two teams exchanged ideas on HP and Education. NJTech_China suggested that our HP should be diversified and deepened. They believed that we should give full play to the advantages of normal universities. They mentioned that the project content can be publicized for young students in school, some primary and secondary students, children and the community. The uniqueness of the project should be demonstrated. At the same time, the two teams discussed some details of the activities and competitions. In terms of rules and evaluation, NJTech_China had rich competition experience. You could obviously feel that NJTech_China was very familiar with the official rules. They had a lot of ideas and breakthroughs, and put forward specific suggestions to our team in the PV production part. Experimental part:We did a series of experiments, using the C16/18 elongase gene from Misgurnus anguillicaudatus they provided for us, to get the results: The effect of C16/18 elongase gene from Misgurnus anguillicaudatus for catalysis on C16:0 is not as good as that of rElo2. In this offline communication, NJTech_China also recommended the C16 / 18 elongase gene from Mortierella alpina to us. We subsequently further applied it to the experiments to look at its catalytic effect on C 16:0. As illustrated in following figure We have learned that the NJTech_China is currently using the FNR transcription factors as an anaerobic-inducible-type of biosensor to regulate the PerR gene. We recommended the vgb promoter to regulated PerR gene, which is most efficiently transcribed under microoxygen conditions and is inhibited at normal oxygen concentrations. Based on the full trust of two sides, we will advance the project toward the common goal. Through this offline exchange meeting, the two teams planned joint activities of relevant schools and planned to jointly host an offline exchange meeting with the theme of education. Through this in-depth communication, our team reflected and sorted out according to the current situation of the project. From the rules, refine the team.At the same time, we planned to jointly hold an offline meetup in August. We discussed many details of the event, such as the proposed invitation team, and began to prepare the venue and souvenirs.

5. Establish Meet Up Cooperatively

August 5, NNU-China 2022 and NJTech_China held Education&Meet Up jointly on the Biomedical Public Technology Service Platform in Jiangbei New Area, Nanjing. Nanjing University (NJU-China), Nanjing Medical University (NJMU-China), Nanjing Agricultural University (NAU-China), Nanjing XinDongfang (NJXDF-CHN), Worldshaper-Nanjing and Worldshaper-NJBIOX also participated in this activity. The 8 teams conducted friendly exchanges and sharing on project initiation, social practice progress and other content. This activity was mainly divided into three parts: lecture on educational methods and skills, exhibition of existing achievements, exchange and discussion. NNU-China 2022 invited Shen Xiaohai, a teacher from the School of Food and Pharmaceutical Engineering of Nanjing Normal University, to share online. Mr. Shen analyzed and explained the following social practice. He mentioned how to do it, the object oriented, the content and effective content collection. All team representatives benefited a lot after listening. On the basis of introducing the contents of the team project, representatives of each team shared the activities of education since the project was carried out. NJTech_China based on the transformation of Clostridium butyricum, team realized the transformation of possible alien soil for future immigrants and provided a new solution. They hosted two summer camps in July and invited senior high school students from Nanjing to start a wonderful experiment trip. Based on the transformation of Clostridium butyricum, NJTech_China has provided a new solution for the transformation of alien soil that may exist in the future immigration. In July, they hosted two summer camps, inviting senior high school students from Nanjing's high schools to start a wonderful experiment trip. NNU-China 2022 was represented by Wang Haoxuan. She introduced the background and content of the project. She proposed to build a new DHA/EPA transportation process based on eggs, contributing to the promotion of human health and food safety in the world. In terms of education, Wang Haoxuan reported on nearly ten activities carried out by NNU-China 2022. These activities are aimed at children, middle school students and the general public. The team produced picture books, brochures and other materials, and carried out community lectures, laboratory open days, etc. These activities were appreciated by everyone. After reporting by each team, members of each team had in-depth exchanges on the progress of other interested teams. They hoped that there would be more in-depth and multifaceted exchanges in the future. Each team had its own advantages. All the teams had friendly exchanges on respective areas of expertise. Finally, the meeting ended in a friendly atmosphere. In this meeting, each team has gained from the exchange. NNU-China 2022 has learned from the experience of other teams and absorbed many new solutions. In the process of continuous improvement, we will maximize the effectiveness of teamwork.

Fig8:Event Posters & Souvenirs

Fig9:Group photo of participants

6. Later Communication & Mutual Provision of Optimization Scheme

On September 11, NJTech_China came to Nanjing Xianlin District for offline communication with us. At this time, the iGEM competition has come to an end. In this exchange process, both sides exchanged experience in wiki production on HP. In terms of experiment:in In this offline communication, we are specially grateful for the recommendation of C16/18 elongase gene from Mortierella alpina NJTech_China in the last communication. After our series of experiments, we proved its great catalytic effect on C16:0. It has been applied to improve the production of ω-3 polyunsaturated fatty acids namely EPA and DHA. In addition, NJTech_China gave specific feedback on the vgb promoter we recommended to them: the transcriptional effect of the vgb promoter was indeed suppressed under normal oxygen conditions, but the inhibitory effect was not obvious. They intend to modify the vgb promoter to enhance its regulatory performance.

1、Introduce the Project and Discuss the Cooperation Plan (early May)

In early May, NJXDF-CHN https://2022.igem.wiki/njxdf-chn/partnership came to Nanjing Normal University. NNU-China 2022 and NJXDF-CHN met for the first time in Tiangong Building. Both teams introduced their projects. We learned that the project theme of NJXDF-CHN is the green sustainable development of phenylethanol. We found that in the experimental part, they also select Yarrowia lipolytica as the chassis. We believed that we could help each other and make progress together in the follow-up experiment.

Fig1:Discuss the cooperation plan

2、Impartment of Molecular cloning technically (May 28)

As NJXDF-CHN is a team of high school students, the foundation of molecular cloning is relatively weak. On May 28,2022, we and members of the NJXDF-CHN team conducted a series of partnership activities for guidance and teaching of genetic engineering and molecular cloning. In the course of the activity, we taught them the basic principles of molecular cloning the skills of primer design and Golden Gate method to construct plasmids. The NJXDF-CHN team members are very grateful for our teaching, and we promise that they will continue to guide them in the subsequent experiments, especially the operation of Yarrowia lipolytica.

Fig2:Impartment of Molecular cloning technology

3、Laboratory Safety June (June 10)

On June 10, our team arranged a laboratory safety manual. We explained the laboratory safety to NJXDF-CHN members, making them familiar with the structure of the laboratory and the use of instruments and equipment.

Fig3:Laboratory safety

4、Offline Communication -- Show Picture Books, Play Plasmid Puzzles and Discuss PV (July 1)

On July 1, NNU-China 2022 and NJXDF-CHN carried out offline communication activity. This time, we mainly had an in-depth exchange on the production of picture books, peripherals and pv. The menbers making picture books and peripheral items in our team showed the production results to NJXDF-CHN. We put forward ideas and suggestions for the project of the other team, as well as details that should be paid attention to during the production process. The two members in charge of pv production in our team explained in detail to NJXDF-CHN about the use of the software we used in pv production, which provided them with the production plan for pv production. NJXDF-CHN also showed us their ideas on the follow-up work during this exchange, which gave us some new ideas.

Fig4:Offline communication

5、Impartment of Yarrowia lipotica genetic manipulation technology (July 13rd-15th)

On July 13rd-15, NNU-China organized an activity to train NJXDF-CHN on Yarrowia lipotica genetic operation. Because we both choose Yarrowia lipolytica as the chassis cell, this is a new type of unconventional oleaginous yeast. NJXDF-CHN has already had a certain understanding on Yarrowia lipolytic, but has no experience in the cultivation and genetic operation of Yarrowia lipolytic. So we spent 3 days to teach NJXDF-CHN team members about the experimental operation techniques of Yarrowia lipolytic, including inoculation, culture, and transformation. NJXDF-CHN team members quickly mastered these skills, and they believed that this activity played a key role in promoting the experiments of their project.

Fig5:Impartment of Yarrowia lipotica genetic manipulation technology

6、Community Preaching (July 21)

On July 21, 2022, NNU-China 2022 and NJXDF-CHN carried out a propaganda activity in Nanjing Wangzilou Community. The activity centered on DHA/EPA eggs and phenylethanol. During the event planning period, we jointly made the event planning plan. We also designed interactive games related to the content of the presentations for the children participating in the activity. During the activity, the two teams gave a series of explanations on DHA/EPA eggs and phenylethanol. Later, we led the children to observe Saccharomyces cerevisiae with a microscope and play interactive games together. Together, we will bring the fun of science to children and plant the seeds of synthetic biology in their minds.

Fig6:Community preaching

7、Gene knockout of Yarrowia lipotica (July-August)

NJXDF-CHN attempted to engineer Yarrowia lipolytica as a competitive platform host to produce 2-PE. To improve 2-PE titer, some competing pathways are needed to be deleted. The major byproduct is analysed to be phenylacetate, which is the oxidized product of phenylacetaldehyde catalyzed by aldehyde dehydrogenases (ALD2 and ALD3). Since NJXDF-CHN has not mastered the gene knockout technology of Yarrowia lipotica, we helped them to knock out these two genes. A marker-free gene knockout method based on Cre-lox recombination system was used as previously reported. And we sequentially knocked out both ALD2 (YALI0D07942g) and ALD3 (YALI0F04444g) in po1fk. The resulting engineering strain was then handed over to the NJXDF-CHN team, and they obtained a very good phenotype on 2-PE production. NJXDF-CHN team believes that the gene knockout work we help them to do is an important step for them to complete the engineering success.

Fig7:Help with gene knockout

8、Publicizing Synthetic Biology in Phoenix Book City (July 24)

On July 24, 2022, NNU-China 2022 and NJXDF-CHN held a publicity campaign on synthetic biology and Omega-3 eggs in Phoenix Book City. In order to adapt to Phoenix Book City, a place with dense flow of people and a wide range of people, we changed our publicity methods. We used fixed booths to display, distributed leaflets, and designed on-site questionnaires to adapt to different age groups on the site. Through the on-site feedback of different groups, we could get more comprehensive public information feedback.

Fig8:Publicizing Synthetic Biology in Phoenix Book City

9、Discuss Wiki and Material Writing (August-September)

In September, we had an offline discussion with the NJXDF-CHN team. We discussed the problems encountered in wiki pages.

Fig9:Discuss Wiki and Material Writing