We decided to engage with two other iGEM teams as partners this year from dry experiments and wet laboratory experiments.
We are very pleased to collaborate with the iGEM team of Fudan University that wanted to develop a rapid detection method for the Candida albicans in resource-limited settings and for point-of-care analysis this year. Both teams focused on the isothermal amplification and simplified detection, so we maintained our partnership during the whole competition season and formed a strong connection that were mutually beneficial to each team. The collaboration with the Worldshaper_Shanghai team was also a valuable asset to our human practice. In this collaboration, we focused on publicizing the iGEM and scientific knowledge of our own project for the general public, which brought a lot of happiness and also made our project meaningful to the public.
The Fudan iGEM team
By chance, we learned that the project of the Fudan iGEM team this year was about the rapid detection of Candida albicans in an environment with limited resources, which was very similar to our project of rapid and laboratory-free detection of virus at room temperature. Thus, their project had aroused our great interest. Our two teams held the first Tencent Meeting in August, 2021 to specifically introduce the projects to each other and explored opportunities for collaboration.
During this meeting of the project introductions and many subsequent online meetings, we provided suggestions and shared ideas to each other's experiments, so we decided to maintain long-term collaborations. We kept the intellectual interactions by holding monthly online communication, but any on-site meeting was still impractical due to the long distance between the two universities and COVID-19 pandemic situation.
Figure 1. Introduction of the project designs to each other.
What the Fudan team brought to our NEFU_China team
We shared our experimental progress and new experimental design every week. Aiming to establish a detection system that is sensitive, specific and convenient in detecting various viruses, we used the M13 phage genelll to test the concept and designed three parts -- RPA to amplify the genelll, single-stranded DNA (ssDNA) displacing to produce ssDNA, RCA to generate multiple G-quadruplex (G4) motifs, which would bind hemin to act as an activated peroxidase to oxidize a substrate, 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS). Then, the color's change of the oxidation process could be visualized very quickly, which provided an instant detection of a virus.
In early September 2021, our ssDNA displacing experiment encountered difficulties. We found that Phi29 protein not only had strand displacement activity, but also possessed 3'-to-5' ssDNA exonucleolytic activity, which could adversely affect the results by degrading the displaced ssDNA. We described this issue to the Fudan team, and they proposed a new method for us that could solve the problem. Klenow, the large fragment of the DNA polymerase I of E. coli, also has strand displacement activity, similar to Phi29, but lacks exonuclease activity. This protein can be well expressed in E.coli. We immediately started to test Klenow and successfully detected its activity in displacing ssDNA. The results are as followings. [Results] for more details.
Figure 2. EMSA to analyze the results of ssDNA displacement
Their suggestion helped us pass through the bottleneck in the ssDNA displacement experiment. We obtained sufficient displaced ssDNA that greatly improved the sensitivity of our detection method. Overall, the partnership of our two teams is significant to our project.
Wet lab partnership
Wet lab partnership is another aspect of our collaboration with Fudan. We think it is necessary to mutually verify the research data, so we carried out collaboration to verify the feasibility of each other's experiments. We sent our RCA system and G4 color reaction system, including the experimental protocol, to the Fudan team, hoping that they could also get positive results with their laboratory reagents and setting.
In the Fudan team laboratory, they still got positive results with their own reagents. The results are as follows (Figure3，4). The experimental group showed green color after the RCA reaction and substrate addition, which suggested that the G4 motif was amplified. The negative controls without either of primer, template, Phi29 or T4 DNA ligase did not show any color change.
Figure 3. G4-mediated color change after RCA
Figure 4. Absorbance of the reaction mix after the reaction The confirmative results obtained by the Fudan team helped us verify the feasibility of our experiments.
The NEFU_China 2021 team sincerely invited all civilian artists to participate in our drawing competition, to contribute to the popularization of the synthetic biology and let more people know this relatively new research field. To let people know about this event, we announced this painting competition to the public through the promotions of our team’s official WeChat and bilibili accounts and other online media, such as Facebook. In addition, we also carried out offline events for the students of our university through poster presentations and other approaches. The Fudan team also actively helped us to publicize our competition to increase its influence, which let us receive more excellent painting works.
Figure 5. The Fudan team publicity moment on their WeChat.
What the NEFU_China team brought to the Fudan team
In August 2021, the Fudan team found that their initially used isothermal amplification method, tHDA, could only marginally show positive results with very weak signal. As a result, it was difficult to combine tHDA with their subsequent LFA design to obtain definitive positive results. This issue could not be addressed after communicating with the customer service staff of the commercial kits that they purchased. When they felt very puzzled and asked for help from many sources to solve the problem, we had an online discussion with their team through the Tencent Meeting platform. We suggested that they should consider to utilize more mature and widely used isothermal amplification methods, such as the loop-mediated isothermal amplification (LAMP). They took the advice and interviewed a professor in Fudan University about the LAMP. Finally, they changed their protocol by replacing tHDA with LAMP to carry out the detection.
Wet lab partnership
The Fudan team aimed to construct an easy, cost-effective and point-of-care detection method of Candida albicans, a conditionally pathogenic strain that can colonize on genital and gastrointestinal mucosa with potential of causing diseases, such as vulvovaginal inflammation. They used E. coli to overexpress the Bst DNA polymerase needed in LAMP reaction. Such an enzyme amplifies a specific DNA sequence of Candida albicans to achieve the purpose of rapid and sensitive detection. Besides, they designed gene circuits with the core parts gp2 and gp5.7 to suppress the background protein translation and increase the purity of the Bst DNA polymerase large fragment produced by E. coli. Through fusing Bst with various types of DNA binding proteins, they hoped to simultaneously reduce the cost of the detection and increase the efficiency.
We helped them to test the feasibility of their products in our lab. The Fudan team shipped the Bst DNA polymerase large fragment produced by their core circuits to us and other reagents necessary in testing their detection protocols. We followed their protocols and got positive results. We also provided feedback to them as target users emulating using their product. We present the detection results (Figure 8,9) and the experimental procedure below.
1.LAMP results were analyzed by agarose gel electrophoresis
Figure 6. Agarose gel electrophoresis analysis of the LAMP results.
2.Testing strip results
Figure 7. Test strip results of the LAMP reaction.
The Worldshaper_Shanghai team
Exchange of molecular biology experiments
At very early stage, we knew that the Worldshaper_Shanghai team is from several high schools in Shanghai, so they might lack basic knowledge of molecular biology experiments and technologies. Considering the difficulty for senior high school students to understand molecular processes, we demonstrated different research experiments to them, including PCR, agarose gel electrophoresis, plasmid construction, protein expression and other experimental knowledge and experience by Peng Jiang, one of the best members in our team. We also trained them many molecular biology techniques and helped them fully prepare for the formal start of their experiments. This process also helped us to consolidate our knowledge and deep understanding of molecular biology.
Figure 8. Molecular biology technology training to the Worldshaper_Shanghai team from a high school.
After we decided our competition projects in middle of May 2021, we held a lot of meetings to discuss and modify each other’s projects, introducing the principles and methods of our projects to each other. Both teams received a lot of comments and suggestions. They praised our sensitive design for testing, but also pointed out the questions about the detection cost, which could still have room to improve.
Figure 9. Project introduction between our team and the Worldshaper_Shanghai.
The Worldshaper_Shanghai team committed to the research of Drosophila related to anti-Alzheimer's drugs. We also carefully listened their presentations and put forward some suggestions.
Figure 10. Project introduction between our team and the Worldshaper_Shanghai.
At the middle of stage, we both started the human practice parts. We had online meetings several times to discuss the ideas about the human practice parts. We shared the experience of our CRISPR conference, the ICII Joint preaching and some other educations to public activities with them.
Figure 11. The online meeting to share experience in human practice activities.
They indicated that they would like to collaborate with us in holding a live publicity, in which we also expected. After several Tencent Meeting discussions, we finally confirmed that the event would be broadcast live through the Bilibili platform, which was finally launched at 4.00 P.M., October 2, 2021. Our two teams together presented the knowledge of the COVID-19 and Alzheimer’s disease.
Figure 12. A member（Xiangxin Li） of our team is preparing the live publicity.
Figure 13. The screenshot for a moment in live publicity.
We publicized the iGEM competition, and let more people learn the popular scientific knowledge related to our project through the live broadcasting. This event greatly increased the influence of the iGEM, making the iGEM not only as a synthetic biology competition, but also an organization enriched with the knowledge of popular science related to everyone’s daily life.
In the final submission phase, the Worldshaper_Shanghai team encountered problems when trying to insert their promotion video into the homepage of their wiki. They did not know the specific codes used to insert the video clip into the website. Immediately, we used the VSCode to help them solve this problem. They sent their original codes to us and we analyzed which step was wrong and the corrected it.
Figure 14. How we helped the Worldshaper_Shanghai team to solve the problem of uploading a video clip.