NEFU was our partner whose program focuses on the point-of-care detection of SARS-Covid19. Both Teams focus on isothermal amplification and the simplicity of detection so we became partners during the whole competition season and formed a strong connection from which we gained mutual benefits from each other.
# How did We Meet
Fudan got in contact with NEFU in April in the Wechat group of Chinese iGEMers.
# Online Meetings (2021.4-2021.8)
We remained a monthly online meeting with NEFU due to the distance and the pandemic.
In April, we shared a research study about isothermal amplification of both sides since both teams entailed isothermal amplification in the project.
In August we held another online meeting to share the design of both teams and we discussed the problems we encountered in the experiment and offered each other advice and encouragement.
Figure 1. Discussing current isothermal amplification methods
Figure 2. introducing project designs to each other
# Inspiring Each Other (2021.8 & 2021.9)
In August, we found that the original choice of isothermal amplification method - tHDA could only get very weak positive results, and the signal was very weak. It was difficult to combine tHDA with our subsequent LFA design to obtain obvious positive results. This issue was not addressed after communicating with the customer service staff about the kits we purchased. When we felt very puzzled and asked for help from many parties to solve the problem, we had an online Tencent discussion with NEFU students, who suggested that we could consider more mature and widely used isothermal amplification methods such as LAMP. We took the advice and interviewed a professor in Fudan about LAMP. Finally, we changed our minds and replace tHDA with LAMP to carry out the detection.
Team NEFU-China aims to establish a detection system that is sensitive, specific, and convenient to detect various viruses. They use the M13 phage general to test the concept and designed three parts--RPA to get amplifies the general, single-stranded DNA displacing to get ssDNA, RCA to get multiple G-quadruplex(G4) motifs which bind hemin that acts as an activated peroxidase to oxidize a substrate, 2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS). Then the color’s change in the oxidation process can be visualized very quickly, which provides instant detection of a virus.
In early September, Team NEFU-China's Single-stranded DNA displacing experiment encountered some difficulties. They found that Phi29 protein not only has strand displacement activity but also has 3 '-to -5' ssDNA exonucleolytic activity, which could adversely affect the results by potentially degrading the displaced ssDNA. They proposed this difficulty to us. After thinking about it, we proposed a new method for them that may solve their difficulties. Klenow, a protein with strand displacement activity as same as Phi29 protein but no exonucleolytic activity, is an E. coli protein, which can be well expressed by E. coli solving the difficulty well.
Our suggestion makes Team NEFU-China break through the bottleneck of the Single-stranded DNA displacing experiment and gets multiple displaced ssDNA making the detection response more sensitive and greatly enhancing the detection effect, which is of great significance to their project.
# Wet Lab Partnership (2021.9-2021.10)
The wet lab was another area where NEFU and Fudan collaborated. The two teams proved the feasibility of the experiment. NEFU used our team’s Bst polymerase to carry out their RCA reaction and compared the efficiency with Phi29--the enzyme they chose in design. Fudan tested the feasibility of the point-of-care detection system by shipping LFA testing strips and (LFA) to NEFU and receive positive results using the exact methods described in the implementation page of Fudan(https://2021.igem.org/Team:Fudan/Implementation). Since Fudan and NEFU both aimed to construct a point-of-care detection platform, it will be better to make sure the test can be carried out in various scenarios.
# Experiments NEFU Carried Out
NEFU helped to test the feasibility of our product in our lab. We shipped testing paper strip and Bst DNA polymerase large fragment produced using our core circuits without purification to them and other reagents entailed in testing the detection protocols. NEFU followed the protocols and got positive detection results and provided feedbacks as target users emulated using their product. Below are the results NEFU got.
Figure 3. Agarose gel electrophoresis results of LAMP
Figure 4. Testing strip results of LAMP
# Experiments Fudan Carried Out
We followed the protocol provided by NEFU and got positive results. The experimental group showed green after the RCA reaction and G4 color reaction, which meant the G4 had been multiply produced. And negative controls that without primer, Template, Phi29, and T4 ligase were all colorless.
positive control experimental group -primer -Tamp -Phi29 -T4 ligase
Figure 5. G4 color reaction results after RCA
Figure 6. Absorbance histogram
# Human Practice Partnership (2021.10)
During the whole year, we helped each other in improving our human practices.
Fudan made two online games to promote synthetic biology to adults and the elderly, and NEFU kindly provided us with their feedback on the trial, which made the game more interesting and smooth.
Figure 7. Screenshot of game page
The NEFU_China 2021 team sincerely invites all civilian artists to participate in our drawing competition, contribute to the popularization of synthetic biology, and let more people know about this relatively new research field. To let more people know about this event, Fudan also actively helped publicize the competition, expanded the influence of the competition.
Figure 8. Team member of Fudan promoting the competition we held on Wechat