ART&WIKI
With BIT-China
With NJMU-China
We believed that prevention and control could be accomplished by engineering Escherichia coli to produce cell-penetrating peptide (CPP) and ERP fusion proteins. These proteins can deliver DNA into cells and therefore could be carried with Pseudomonas syringae pv. Actinidiae carrier of the lysin gene PN09-89 was delivered into the pathogen, causing it to self-lysing to death. However, friends of NJMU-China believed that CPP had the problem of low penetration efficiency, and might have an impact on E. coli itself, and there were certain limitations, too. In our intra-team verification discussions, we did find that there was such a limitation problem, so we decided to abandon this project.
Figure 1. First project of NAU-CHINA.
Uridine is an intermediate in antiviral and antitumor drugs and it has extremely important medical value. We hoped to build a co-cropping system with Bacillus subtilis as the chassis to synthesize uridine. We concluded and divided the synthesis of uridine into two modules. The first module was responsible for the conversion of raw materials to whey nucleotides, and the second module converted whey nucleotides into uridine and efficiently transported them out of the cells, reducing the metabolic pressure on the cells of each module.
NJMU-China proposed that this co-cropping system may disrupt the gene expression and even basic living of the bacteria. What’s worse, the transfer of intermediate products between the two engineered bacteria modules would affect the efficiency of co-crop systems. Therefore, after discussion within the team, we decided to temporarily suspend this idea.
Figure 2. Second project of NAU-CHINA.
The project was about diagnostic testing. NAU-China team members paid attention to the lethal liver disease around the world. Bile acid was an important indicator of liver disease, so we intended to take advantage of the amount of bile acid for targeted patients to detect liver-related diseases at home conveniently.
In our discussion with NJMU-China, they briefly explained various types of liver disease to us, hoping to help us learn more about backgrounds, and asked whether there is any concrete data that can support the change of bile acid content in the urine after liver disease.
This question was crucial to the design of our topic. Numerous studies have investigated people with Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Nonalcoholic Steatohepatitis (NASH), Alcoholic Liver Disease (ALD) and Primary Bile Cirrhosis (PBC). The data indicated that there are many differences in bile acids between some patients and normal populations. The absolute concentration and its composition are considered to be the most obvious differences. It was evident that bile acid concentrations were much higher in the five etiological groups than in the healthy control group, and the marker CDCA which we selected was Significant differences between the five etiological groups and the healthy control group (Table 1).
Table 1. Bile acid levels in the control group and 5 pathological groups (mean ±sd)
| Variable | Control (n=153) | HBV (n=400) | HCV (n=214) | NASH (n=214) | ALD (n=37) | PBC (n=57) |
|---|---|---|---|---|---|---|
| CDCA* | 134.55 ± 196.45 | 197.89 ± 293.33 | 339.67 ± 318.98 | 488.49 ± 602.05 | 913.44 ± 1286.88 | 478.31 ± 732.48 |
At that time, NJMU-China explained some background knowledge about autism spectrum disorder and gave a general introduction to their project this year. They were trying to solve multiple problems with autism treatment at the same time. Taking the cost and the accuracy of the experiment into consideration, we suggested that they could focus on thinking about a certain aspect of autism treatment and refining it. (For more information, please click here)
Figure 3. Project explained by NJMU-China.
In 2022, we have frequently exchanged ideas with two outstanding iGEM teams: BIT-China and NJMU-China, on different topics. We also have routine communication between the sub-groups within our own team.
Thanks to the escort of our Art and Wiki members, our ideas of experiments and human practices came to reality. At the beginning of June, 2022, we explained the design of our project to the Wiki members clearly to enable them to build the web platform more smoothly and quickly, after the initial improvements on the experimental topic. In addition, we guided the Art members to translate some obscure expressions into image-specific ways so as to better illustrate them.
In June, the Art members completed the design the mascot catering to the topic this year: small liver. At ICII in July, they produced poster brochures and designed team logo T-shirts. Our PV of this Year was also completed after collaborations with the art team and got freshly released in August. Throughout the project, our artists designed several theme products to serve Human Practices activities.
It was worth mentioning that our Art and Wiki members had actively conducted communication and exchanged in the process of building the web pages in order to design a unique layout and easy-to-understand form. All of these interactions enhanced the unity of our team, promoting harmony within the team and future development.
NAU-CHINA and BIT-China cooperated in-depth in many dimensions. Adhering to the principle of cooperation in different aspects and multiple points, we had deep communication on the basis of mutual strengths and weaknesses. The collaborating highlights were recorded below:
To promote our projects, we displayed educational videos to each other respectively. NAU-CHINA reposted BIT-China's three-minute video in the team's WeChat group, which shared activities of scientific knowledge, and these videos were highly praised by students who were interested in synthetic biology on campus. BIT-China also reposted and promoted our original sitcom "Liver Troubles", which was produced for the project. The lively and interesting content was recognized by our partners.
Figure 1. Appraise of BIT-China after watching video.
In addition, NAU-CHINA held a campus lecture in October. We registered a WeChat official account to promote the warm-up activity before the lecture, which attracted a large number of students who are interested in synthetic biology and iGEM to participate in. During the lecture, in addition to introduction to synthetic biology, iGEM as well as our project, we briefly illustrated the posters and the project of BIT-China, which were deeply thought through and praised by attendees.
Figure 2. On-site audience for Campus lecture.
Figure 3. NAU-CHINA member explained project.
Finally, we would like to thank BIT-China for introducing our project posters at the iGEM Synthetic Biology Mini Jamboree in Beijing in October. In this carnival and biological cross-border art exhibition, BIT-China distributed our team's cultural and creative theme products designed by the two teams to many local iGEMers. Though we are a thousand kilometers apart, we felt the enthusiasm of the BIT-China, which gave us the motivation to strive forward.
Figure 4. Visitors with poster of NAU-CHINA .
During the competition, we had two in-depth discussions with BIT-China about the mathematical model. In the first meeting in June, we intended to apply the τ-leaping algorithm to simulate the genetic circuit to complete the estimation of the protein amount under specific conditions, but we did not get the exact DNA transcription rate of FXR and RXR at that time.
In response to this problem, BIT-China provided more precise parameters of the plasmid vectors and proteins for us, which helped us complete the more accurate calculation of the product quantity. When we introduced molecular dynamics simulation, BIT-China believed that our thinking was clear and complete, and we offered suggestions and references for their model, which laid the foundation for one of their models.
Figure 5. Online meeting with BIT-China.
In the second discussion from August to September, we had not finished the complete code for the model for predicting protein structure with support vector machines (SVM). With the help of BIT-CHINA, we established a complete SVM model for predicting protein structure. During the process of communication, we determined the search and import of the required training set. We also brought the formula and parameters of the dynamic simulation software to BIT-China to help them further improve the construction of their model.
During these two online meetings, we carefully solved the questions raised by BIT-CHINA, along with jointly learning mathematical simulation software, such as Molecular Dynamics Simulation, τ-leaping, Support Vector Machine, and flexibly applying them to our respective projects. We also conducted a simple exchange of the results obtained from the experiment and kept close communication until the end of the project.
The Wet Lab is an important part of the whole collaboration. We mainly asked BIT-China more questions about experimental technology. The following is a timeline of experimental cooperation:
During this period, our cooperation mainly focused on introducing and learning of our
experimental designs respectively.(Please click here for details)
From June to July
Based on the review of previous literature, we decided to build a system with low-cost maltodextrin as energy source, but some reagents were difficult to obtain, which limited the development of our experiments. After a brief discussion with BIT-China, we got their suggestions that bacterial extracts could be used and optimized by adding substances such as lactose, making it an extremely low-cost cell-free reaction system. Then we found that the bacterial extracts had already contained tRNA, NAD, cAMP and other substances in the system with no exogenous addition required. As a result, the problem of improving the system can be solved more easily.
Figure 6. Experimental notebook of NAU-CHINA members.
As a partner, we were very willing to help BIT-China in the brainstorming stage to find out the advantages and disadvantages of multiple alternative project ideas and put forward our ideas about the feasibility of those ideas. (For more information, please click here)
After months of experiments, we had figured out a clearer experimental direction and
some pre-experimental results.(Please click here for details)
From July to August
However, we found that the dimerization of ddRFP did not show the expected fluorescence after binding. As to this problem, we asked BIT-China for advice. They suggested that chances were that the protein concentration in the bacteria was not high enough, or that CDCA was more difficult to enter the cell and did not reach the right concentration to activate the binding of the two receptors.
Therefore, it was suggested that we could try to purify and concentrate the protein. Their tips provided us with new ideas that the linker between ddRFP and receptor might bring obstacles. If the linker was improperly selected, the distance of the two proteins may be so close that the misfolding of FXR and ddRFPA1 would occur, and this phenomenon might further affect the structure of the ddRFP chromophore, preventing red light from appearing.
Figure 7. The scenes NAU-CHINA discussed.
During July and early August, due to the epidemic, BIT-China had not yet returned to laboratory to conduct experiments. Therefore, we mostly recorded biological experimental skills, experimental operations, etc. to them through video. (For more information, please click here)
Later on our experiment, the exchanges between our two teams were more inclined to
introduce the experimental results and improve the details of the final period.(Please
click here for details)
From August to September
We helped BIT-China to improve the solution for the selection of promoters and the creation of a low-oxygen environment for experiments. Meanwhile, for the fluorescence problem that plagued us in the mid-term, we got the answer from the other team's prompt and our own thinking.
We shared the verification method immediately. Firstly, the two parts of ddRFP were connected in series and expressed constitutively, and then the result did not appear in red light. We further changed for the T7 promoter to increase the concentration of the protein. At this time, a brighter red color appeared, which proved that the appearance of fluorescence may be related to the expression of the protein. Therefore, it was believed that the linker had little effect on these two proteins. BIT-China agreed and praised our verification steps.
Figure 8. Online meeting bewteen BIT-China and NAU-CHINA.
Through more than five months of experimental communication, we were being inspired mutually, and these inspirations had influenced the advancement of our projects respectively. (For more information, please click here)
The completion of the above-mentioned cooperation was also inseparable from the technical cooperation support of the art team. Together with BIT-China, we designed the theme image of the collaboration, with figurative liver and Escherichia coli posing diverse and friendly gestures. These theme images were redesigned into postcards for mailing and on masks for daily wearing.
In addition, the help from our art friends was undoubtedly an important part of our successful completion of the ICII. Art members from BIT-China participated in the design of some ICII brochures and part of the editing of the final summary video. The beautified brochure attracted a large number of students to participate in the promotion event of other teams in the school, and the simple and generous summary video was highly praised by all teams, too.
Figure 9. Cultural theme products about marks and postcards.
Figure 10. Mascot between BIT-China and NAU-CHINA.
Since we met and interacted with the NJMU-China in the last season, our two teams have become friends now. At that time, we were both interested in each other's subjects, and coincidentally, at the end of 2021, we both indicated that we had the idea of doing a diagnostic track in the following year. Therefore, during the brainstorming stage in March, we established a close partnership and decided to shape a form of cooperation with the wet lab as the core and radiated outward throughout the season this year.
In April, our team members were in the brainstorming stage, and we came up with three alternatives. We evaluated these three projects and tried to figure out the one to choose. At that time, we were grateful to the students with NJMU-China for helping us analyze the advantages and disadvantages of the three topic ideas. Their suggestions provided us with the direction of improvement related to the project and enhanced the integrity. The following are NJMU-China questions and suggestions for our three alternatives: (Please click bulbs for details)
After more than a month of design and preparation for operation, we had a discussion with friends from NJMU-China in June to improve the details of the experimental design.
Figure 4. Online meetup with NJMU-China.
NJMU-China provided us with valuable advice on how to present the system in a cell-free system. Originally, our original idea was to use the lyophilized bacteria as the final product. While NJMU-China suggested that the cell-free system was relatively safe for fewer safety concerns like bioleakage, and could have higher detection efficiency when used as a biosensor. Fortunately, this can avoid the problem of biomarkers entering the engineering bacteria and affecting the test results, which is conducive to the detection of the more trace biomarker in this topic: bile acid.
NJMU-China also reminded us that people living in different geographical regions might have different levels of bile acid in their urine. In response to this problem, we retrieved a large number of literature, and found that the content of bile acid in the urine of liver disease patients in East China, represented by Shanghai, China, was significantly higher than that of healthy people, so we decided to take the concentration of bile acid in the urine of patients in this region as the benchmark index and East China as the first market.
In July, experiments in both teams were carried out in an orderly fashion. We decided to focus on sharing the digital modules of the two teams with each other. In the seminar, we presented details about the Support Vector Machine (SVM) to predict the secondary structure of the protein, including the way how amino acids were encoded and the specific classification of protein secondary structures. These contents provided a perspective for NJMU-China to think about the construction of protein 3D models.
In addition, we also exchanged about the methods of Gromacs, such as the choice of force field, various calculation formulas, energy minimization, etc. Receiving suggestions from discussions with NJMU-China, we gained a deeper understanding of the programming of Gromacs, which also solved some difficulties in force field selection and enriched our knowledge system.
Figure 5. Model meetup with NJMU-China.
After a period of online interaction, we could not wait to meet offline to communicate, and promote cooperation and friendship. Since we were all in Nanjing, Jiangsu Province, there was not so much trouble to go offline. Eventually, at the beginning of August, we had an offline exchange in a tea room.
Figure 6. Offline meetup with NJMU-China.
As usual, we briefed the recent experimental progress and the problems recently encountered. At the meeting, NJMU-China asked us when our product requires the user to collect urine. After that, we realized that the concentration of bile acids in the urine could be affected by diet, activity and medically. Morning urine was more frequently used for laboratory examination, because the concentration of relevant components in the morning urine was relatively high and stable, ensuring a more accurate detection of disease.
At the same time, we found that in NJMU-China’s design, their chelate might have the possibility of being affected by pH changes in terms of preventing the accumulation of heavy metal ions, which would eventually affect the state of bacteria after lysis. What’s more, whether the produced toxic proteins may leak into the environment was another critical concern.
Our discussions about these problems helped the two teams during the bottleneck stage of the experiment.
Figure 7. Members of BIT-China and NAU-CHINA in this meeting.
In this hard-won in-person exchange, we learned about the activities held by the two teams and planned the future scheme in the next few months in the Human Practices section. We also asked how to contact doctors in hospitals effectively to assess safety and ethical concerns in our experiments. These pieces of advice contributed greatly to our integrated Human Practices part.
We cherished this meeting very much. After the warm-up activities, we decided to enhance our cooperation and friendship through a variety of games we designed, including Synthetic Biology Draw & Guess, Uno and other board games to add a joyful atmosphere.
Figure 8. Members of NAU-CHINA.
Figure 9. NJMU-China and NAU-CHINA.
In addition, we also participated in the Education Meetup organized by NNU-China and NJtech-China in August. At that event, we highlighted the contents and innovative ideas that we decided to complete in the Human Practices section, and exchanged problems and experiences encountered in carrying out those activities. We thought highly of other teams’ activities and learned a lot.
Figure 10. Education meetup.
As partners, we thought we should design a couple (CP) image to memorize the friendly relationship, so the Art members of our two teams drew some animated images this year to integrate with the logo of two teams in order to show a great web image.
Figure 11. Couple image with NJMU-China.
Sang, Chao, et al. "Bile acid profiles are distinct among patients with different etiologies of chronic liver disease." Journal of proteome research 20.5 (2021): 2340-2351.
