Summary

In the circumstances of the continuous spread of the epidemic, this year, 2021, our team met a group of excellent partners through online communication and offline visits. From field visits and surveys to waste treatment plants, the overcoming of technical problems about Pichia pastoris and CRISPR/Cas, to human practice, we have been supporting each other all the way through the long season, and now we hand in a satisfactory answer.

Partner 1: Tongji_China

—— The start of the PEDe.

1. Collaborative work at different stages.

We have been in contact with Tongji_China for many seasons. At the beginning of this season in April 2021, we organized an online project determination meeting to discuss that for this season. At this meeting, we discussed with

Tongji_China that the cities where the two teams are located both have begun to implement the garbage sorting policy. And we all noticed that the messy collection of parcel waste in the Cai Niao Courier Station on campus. So, we decided to conduct further research on this.

In May, Tongji_China came to NanjinCPU_CHINA Team-Partnership

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Fig 1. Fisrt offline meeting

g to communicate with CPU offline. In this offline communication, Tongji_China shared their project design with us. They hope to use sulfoquinone oxidoreductase (SQR) and Ks-Amt to detect hydrogen sulfide and ammonia, and use AQR, superoxide dismutase (SOD) , ArpBA and SAT are expected to gradually degrade hydrogen sulfide gas to mask the stench produced by wet garbage.

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Fig 2. Communication in both team

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Fig 3. Academic communication

At the meeting, Tongji_China proposed a problem that the secreted expression of SQR and SOD may have low trans-membrane rate, while the way of lysing engineering bacteria to obtain enzymes they produced may face the problem that enzymes are gradated by intracellular proteases. In this regard, our team proposed two solutions respectively: ①We can construct a signal peptide library and screen out the signal peptide with the highest efficiency to assist the target protein with exocytosis, T--CPU_CHINA--Education--Figure 3avoid the encounter between the protease and the target protein as well. ②It can be considered to add protease inhibitor or EDTA to inhibit the protease activity in the process of lysing E. coli.

At the same time, Tongji_China shared the design ideas of the questionnaire with us, hoping to help us optimize the questionnaire for plastic pollution treatment.

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Fig 4. Tokens exchange

In June 2021, Tongji_China and us both were preparing a trial talk of meetup, during which we revised the speech and slides for each other.

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Fig 5. Speech Preparation

In July, with the help of Tongji_China, we completed the synthetic biology publicity and education project "INTO CHINA, INTO IGEM" in

In August, due to a new outbreak of COVID-19, we conducted a site visit to the Jiaozishan Waste Treatment Plant for Tongji_China

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Fig 6. Assisted interviw.

and provided Tongji_China with details of the wet waste treatment methods of the existing waste plant.

2. Benefit from each other.

Tongji_China helped CPU_CHINA:

With the help of Tongji_China, we completed the design of a questionnaire about plastic pollution at the beginning of our project.

3. Working together.

With the cooperation of both teams, we completed the project background research, final project design and polished the speech and slides in multiple presentations. In addition, in education work, we completed the "ICII" project together.

Partner2: UESTC-China

—— It starts with pichia pastoris

Collaborative work at different stages.

At the beginning of this season, April, we met UESTC-China in the iGEM communication community of China area and discussed the project design. In this meeting, we both focused on the issue of environmental protection, hoping to solve the pollution problems in our life.

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Fig 7. Start of our partner journary

After our team confirmed the use of manganese peroxidase to degrade polyethylene, how to choose the bottom bacteria became a difficult problem. In May, we held an online meeting with UESTC-China in order to seek advice from UESTC-China on this issue because UESTC has rich experience in yeast. With their help, our team compared the advantages and disadvantages of Saccharomyces cerevisiae and Pichia pastoris for secreting and expressing highly glycosylated MnP, and finally decided to use Pichia pastoris GS115 for protein expression.

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Fig 8. UESTC-China help CPU_CHINA with choosing of chassis--Pichia Pastoris GS115.

In June, after the trial started, we exchanged the progress of the project for the first time. Discussed and solved the problems of bacteria contamination and PCR pretreatment encountered during the experiment. In addition, the two teams also discussed how to use deep learning and machine learning for directed evolution of enzymes.

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Fig 9. Design of plasmid be used to construct single point mutant.

In July, we jointly formulated a cultural and creative exchange plan, and deeply understood the design concepts of each other's projects, and initially made the work plan of the two teams in popular science education.

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Fig 10 Exchange of our culture-- Good from CPU_CHINA

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Fig 11 Exchange of our culture-- Good from UESTC-China

In August, we assisted UESTC-China in holding the Yeast Meetup, shared experience with the participating teams, and finally completed the writing of the "Yeast Handbook".

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Fig 12. The cover of Yeast Handbook

In September, with the help of UESTC-China, we jointly completed the "ICII" publicity and education work. In October, with the assistance of UESTC-China, we contacted with teacher Jia Fang from Chengdu Yeast Institute and teacher Pei Xiaoqiong from Sichuan University, and discovered common problems in the purification experiments of the His-Tag-labeled protein expressed in Pichia pastoris.

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Fig 13. Supply of the positive sample.

2. Benefit from each other.

UESTC-China helped CPU_CHINA:

UESTC-China helped us determine the use of the Pichia pastoris expression system, guided our team to improve the quality of competent yeast, and assisted us in interviews with professionals about Pichia pastoris . They also helped us test the wild-type Pichia pastoris we used and sent us the reagents needed to complete the new enzyme activity determination method.

CPU_CHINA helped UESTC-China:

We sent dCas9-SpyCatcher protein with His-Tag tag to UESTC-China, and provided a His-Tag tag positive control sample for UESCTA-China.

3. Working together:

We jointly completed the Yeast Meetup meeting and communication work, and collaborated to complete the writing of the Yeast Handbook.

Partner 3: Tianjin

—— Partner brought by CRISPR/Cas technology

1.Collaborative work at different stages.

In April, Tianjin provided us with the idea of introducing CRISPR/dCas9 technology to fix proteins.

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Fig 14. Meet with Tianjin

In May, we discussed with Tianjin about the method of constructing plasmids in Saccharomyces cerevisiae, which served as a reference for us to choose whether Saccharomyces cerevisiae or Pichia pastoris for experiments.

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Fig 15. Comparation between Saccharomyces cerevisiae and Pichia pastoris

In June, we assisted Tianjin to complete a questionnaire survey on the prospects of chromosome-free Saccharomyces cerevisiae.

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Fig 16. Assist Tianjin with interview offline

In July, we participated in the CRISPR/Cas conference held in Tianjin. At the meeting, we discussed the problem of protein expression of both teams and provided practical solutions.

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Fig 17. CRISPR/Cas conference

In August, we interviewed Ren Kejing from the laboratory of China Pharmaceutical University for Tianjin to provide suggestions on CRISPR/Cas technology for yeast chromosome cutting.

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Fig 18. Interviewing for Tianjin

In September, Tianjin provided us with a method that we can use chromosome-free cells to express AAO, the protein Pichia pastoris failed to express.

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Fig 19. Experiment method provided by Tianjin

2. Benefit from each other.

Tianjin helped CPU_CHINA:

Tianjin provided us with ideas and technical references for using CRISPR/dCas9 technology to assemble molecular machines. And Tianjin provided us with an experimental protocol for using their yeast to express AAO.

CPU_CHINA helped Tianjin:

We helped Tianjin design the half-life detection experiment of modified GFP. We also assisted Tianjin to interview researchers related to CRISPR/Cas technology.

3. Working together.

In the season of 2021, Tianjin and us helped each other to solve the problems in the use of CRISPR/Cas related technologies, co-authored CRISPR APPLICATION GUIDELINES, and cooperated to complete the "ICII" publicity and education project.