Full contributions to future teams
Our team has made contributions to the iGEM community in all aspects. And this is the abstract.
- For education, we offer popular biology science courses for the general public, paying attention to the elderly who we ignored previously and promoting educational equity.
- For human practice and entrepreneurship, we promoted our project's progress through communications and eventually formed a complete business plan. Once we gain investments and successfully develop the product, we can supply the elderly with a low-priced and useful calcium supplement product. As a typical case of the combination of scientific research, teaching, and industrialization driven by iGEM, our experience serves as guidance on how undergraduates play their roles in their projects.
- For modeling, we improved the quorum-sensing model of the existing team, using the Gillespie algorithm random simulation to simulate the physiological processes at macro and micro levels and explain the dynamic stability of the antimicrobial peptide. For the Kill Switch model, we improved the logistic equation based on the previous experimental data, adjusted the promoter properties according to literature, and made sensitivity analysis. Our adaptation can be applied to future teams.
- For safety, we surveyed the existing kill switches in the literature and modeled key properties of the cold triggered kill switch, providing an experimental plan for further use.
- For basic parts and composite parts: (1)We combined scattered information in the literature and created our own CaAP (calcium absorption peptide). We showed it could be secreted and has the ability to bind calcium. Then we had it expressed controllably in E.coli to help intestinal cells absorb calcium. (2) We increase the secretion of antimicrobial peptides by adopting a relatively low expression of antimicrobial peptides (mcbA-D) and high expression of the antimicrobial peptide immune portion (mcbE-G), which makes our quorum system more efficient.
- For parts collection, we provided the complete set of plasmids we used to optimize circuits for expressing probiotic secreted proteins. Out experience and results were carefully documented on the Parts Registry, which makes it easy for others to use. Our contribution shows that we are committed to putting ideas of synthetic biology into practice, including engineering and knowledge sharing.
- For measurements, We developed a method to measure blood calcium. We also made simple hardware using LED lamp tubes and mobile phone software, hoping to design a more user-friendly detector for the elderly.
Education
We realized that understanding of biological knowledge varies among different groups of people, so we decided to create new access to basic knowledge for them. We regularly make brief and easy-to-understand biological online audio courses, including audio and text with illustrations, starting from the most basic macromolecules concept. Then we put them on platforms like our WeChat official account. This allows people who have never been exposed to synthetic biology before, even children and the elderly who were ignored before, to have a preliminary understanding of what we can do through synthetic biology. You can read our Education Page for more details.
These popular science materials are available to everyone. They can be released and applied to the public-facing popular science propaganda in our region so that the public and us can reach a consensus on the value of synthetic biology. The sharing of resources also enables more people to participate in our work, conducive to promoting social education equity and development.
This contribution was mainly made by our team member Yao Yiyang. You can go to our Team Page to learn more about our team members.
Entrepreneurship
We promoted our project's progress through communications and eventually formed a mature business plan for at least five years. We hope to gain multiple rounds of investments, successfully develop the product and get official approval. Our ultimate goal is to put it into the market. At that time, we can supply the elderly with a low-priced and useful calcium supplement product.
Our commercial projects is a successful combination of scientific research, teaching, and industrialization driven by iGEM. We sincerely hope that our experience can serve as guidance on how undergraduates play their roles in their projects, promoting the transformation of scientific research results into commercial products.
This contribution was mainly made by our team member Mo Tianqi. You can go to our Team Page to learn more about our team members.
Modeling
We have established two models: quorum sensing process and Kill Switch.
Quorum sensing
In the quorum sensing section, we used the Gillespie algorithm in random simulation to simulate t macro and micro levels' physiological functions. It expresses luxI and luxR genes at the micro level and produces dimers before the population reaches a specific size. At the macro level, Escherichia coli keeps proliferating, excreting antimicrobial peptides, and colonizing the intestinal tract during this process. When the population number is sufficiently large, the short calc-absorbing peptide and the regulation system begin to express, while the antimicrobial peptide is expressed but cannot be discharged so that the bacterial number can achieve dynamic stability. We refer to the model and data of 19-Fudan Team and use the random process to describe it more clearly to model the quorum sensing process in stages and levels. Remarkably, we explained the dynamic stability of McbA.
Kill switch
In the kill switch section, we use 19-BNU Team's data. We improved the logistic equation, adjusted the promoter properties and made a sensitivity analysis. We can see good results through it.
These models confirmed the feasibility of these two modules to a certain extent and provided valuable references for future teams who are interested in this. Also, we showed the process of applying the models of previous teams to our new ones. We hope this can promote the sharing of inspiration and creativity in the iGEM community.
This contribution is mainly made by our team member Jiang Xiaohui. You can go to our Team Page to learn more about team members.
Kill switch
We have known that the environment-friendly kill switch is a genetic circuit that promotes the death of the engineered bacteria when they leave the environment where the designer expects it to work. In the intestine, a probiotic kill switch is frequently used to prevent the modified gene from leaking into the environment when probiotics are released.
New information from literature
![contribution of BBa_K3036004](https://static.igem.org/mediawiki/2020/7/71/T--Fudan--contribution_RelEB2.png)
Based on last year's team, we have added new information learned from the literature that needs attention to RelE/RelB toxin/antitoxin system, which makes the information source more specific and reliable. We found information about potential pathogenicity of RelE, chromosomally encoded RelE/RelB in gut microbiota, and homonymic "Relb" . We regarded these as a must-know for iGEM teams focusing on intestinal probiotics and attempting to use these parts.
Improvements and experimental proof
![contribution of BBa_K3036004](https://static.igem.org/mediawiki/2020/e/ee/T--Fudan--contribution_RelEB1.png)
![contribution of BBa_K3036004](https://static.igem.org/mediawiki/2020/8/89/T--Fudan--contribution_RelEB3.png)
The kill switch in our project this year applies the toxin-antitoxin system. The toxin relE is constantly expressed, while the antitoxin relB is expressed only at body temperature(37℃), which protects the engineered bacteria from toxin. When the environment temperature is lower than 37℃, the antitoxin expression decreases, and the engineered bacteria die.
This kill switch has significant advantages compared to 19-Fudan Team. Last year their kill switch reaches the best killing effect at 40°C instead of 37℃, not suitable for our goal. But this year we make it. It allows the bacteria to survive at 37℃ but suicide when the temperature decrease, which is more suitable for working in the internal environment of the human body. Moreover, last year they had to express a variety of proteins to complete the process of temperature control, which caused the bacteria survival burden heavier. Fortunately, now our kill switch contains fewer essential proteins,helping the bacterial flora stably express genes we insert.
Besides, our kill switch has a more sensitive temperature response and higher efficiency than 19-BNU Team, since we change the temperature sensing riboswitch from RNA thermometer FourU to RNA thermometer NoChill-06.
Due to the impact of the epidemic, we have not been able to conduct a confirmatory experiment, but we still provide an experimental protocol. You can find this in our Experiment . At the same time, we used the original data from 19BNU-China for modeling verification. More specific information is showed on our Modeling Page. These above proves that the kill switch is theoretically reliable and can be experimentally verified. You can read the Engineering Page for more details.
A summary of Kill Switches
We also shared more valuable information based on 16-Marlborough Team's kill switch database. We traversed mostly used methods of kill switches in intestinal probiotics designed by the past iGEM teams. We summarized them into two tables, in which we analyzed trigger conditions and the mechanism of existing kill switches. For parts of resemble mechanism, we only listed the latest and most efficient ones.
Table 1. summary of the mostly used killer system in intestinal probiotics project. More “+” represents higher efficiency.
Killer System | Advantages | Disadvantages | Parts | Efficiency | Comments |
---|---|---|---|---|---|
Toxin | easy, direct | The leakage of toxin may cause engineered E. coli lysing in the body and interrupting native flora. | MazF cas3 Phi 174 Gene E protein | +++ | Expression of toxins alone have been shown to help develop bacterial persistence [2]. |
Toxin/antitoxin system | stable, flexible | / | MazF/MazE RelE/RelB | ++++ | TA systems can stabilize plasmids and facilitate rapid adaptation of gut without negative effect on fitness of the human host. [1] |
Vital gene knock out | efficient | non-universal, time-consuming for testification. | can gene knock out Deactivating Exendin-4 expression system by cas3 | ++++ | Note 1 |
Note 1: The method of can gene knock out by last year’s grand prize winner is ingenious and effective, unfortunately, it can be applied to our project. It is reported that carbonic anhydrase, expression product of can gene, influence the release of PTH, leading to complex effects on bone remodeling. Carbonic anhydrase deficiency is manifested by osteopetrosis in human.[2] Although no concrete evidence has been found to show the relevance between carbonic anhydrase deficiency in intestinal microbiota and osteopetrosis, we decided to give up this method to avoid potential problems.
Table 2. summary of the mostly used response system in intestinal probiotics project. More “+” represents higher efficiency and more “-” represents stronger killing power.
Physical Parameters | Response System | Sensitivity | Killing power | Parts | Comments |
---|---|---|---|---|---|
Temperature (cold shock) | Cold-acting promoter | ++ | --- | PcspA | Working efficiently only below the room temperature (20℃). |
cI repressor system | +++ | --- | cI repressor lambda cI regulated promoter TCI38 TCI | Kill bacteria efficiently only when reaching 40℃, so it is not appropriate for our project that expect E. coli to survive at that temperature. Moreover, it requires to express two types of protein which imposes more burden to the bacteria. | |
RNA thermometer | +++++ | ---- | FourU NoChill-06 | Appropriate reaction temperature range, high sensitivity and high efficiency. | Concentration of essential nutrients in the intestine | glucose-sensitive promoter | ++ | --- | rpoH P5 PT-αCRP | Glucose starvation promoter (BBa_K3142012) initiate transcription efficiently with concentration of glucose lower than 0.05%. The concentration of glucose in small intestine will reduce to 0.05% two hours after dinner and limit the survive of E. coli. |
Fatty acid responsive repression system | + | -- | Fatty acid responsive repression system | Metabolites as responding factors, are greatly affected by food intake. | Other different conditions | N-Acetyl-Glucosamin-6 phosphate regulated repression system | + | -- | N-Acetyl-Glucosamin-6 phosphate regulated repression system | It is not very efficient. |
CO2 concentration | ++ | ---- | can gene knock out | Note 1 | |
light condition | + | - | killer red | It is of very low efficiency and the demand of light illumination greatly limited its application contexts. |
This summary will help future teams understand the killing effects and application scope of various kill switches. They could select the appropriate kill switch according to their own needs to achieve experimental safety.