Full contributions to future teams
Updated on 2020-11-22: we won the Gold Medal.
Our team has made contributions to the iGEM community in all aspects. And here is the list.
- For education, we offer popular biological science courses for the general public, paying attention to the elderly who were ignored previously. We promoted and are promoting educational equity.
- For human practice and entrepreneurship, we improved our project's progress through communications and eventually formed a complete business plan. Once we secured investments and successfully developed 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 the product development.
- For modeling, we improved the quorum-sensing model from the past, using the Gillespie algorithm random simulation to simulate the physiological processes at macro and micro levels. We explained 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 approach can be generlized and used by 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 (mcbABCD) and high expression of the antimicrobial peptide immune portion (mcbEFG), which improved our quorum system efficiency. - For parts collection, we provided a complete set of plasmids we used to optimize circuits for expressing probiotic secreted proteins. Our experiences 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 measurement, we developed a method to measure blood calcium. We also made simple hardware using low-cost LED tubes and an existing mobile phone software, hoping to design a 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 WeChat, Ximalaya, etc. 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 /Team:Fudan/Education 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, collectively to promoting social education equity and development.
This contribution was mainly made by our team member Yiyang. You can go to our Team page to learn more about her.
Entrepreneurship
We promoted our project's progress through communications and eventually formed a complete business plan for the next five years. We hope to gain multiple rounds of investments, successfully developed the product and get official approval for public sale over the counter. Our ultimate goal is to make it reach the elderly in the underdeveloped countries. At that time, we can supply the elderly all over the world with a low-priced and sustainable 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 the project, promoting the transformation of scientific research results into commercial products.
This contribution was mainly made by our team member Tianqi. You can go to our Team page to learn more about her.
Modeling
We have established two models: quorum sensing process model and kill switch model.
Quorum sensing
In the quorum sensing model, we used the Gillespie algorithm in random simulation to simulate the 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 calcium absorbing peptide (CaAP) 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 19Fudan Team and use the random process to describe it more clearly. We modeled the quorum sensing process in various stages and levels. Remarkably, we explained the dynamic stability of McbA.
Kill switch
In the kill switch model, we use 19BNU Team's data. We improved the logistic equation, adjusted the promoter properties and made a sensitivity analysis. We see better results through our effects.
Our models confirmed the feasibility of our design to a certain extent and provided valuable references for future teams who are interested in this. Also, we showed the process of adapting the models of previous teams to our new ones, and improving them. We hope this can promote the sharing of inspiration and creativity in the iGEM community.
This contribution is mainly made by our team member Xiaohui. You can go to our Team page to learn more about her.
Improved safety with a kill switch
An 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
Built on last year's work, we have added new information learned from the literature that needs attention to RelE/RelB toxin/antitoxin system, which makes the information on the Registry page 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 plans
The kill switch in our project this year applies the toxin/antitoxin system. The toxin is constantly expressed, while the antitoxin 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 the one proposed by 19Fudan. Last year, their kill switch reaches the best killing effect at 40°C instead of 37℃, not suitable for intestinal usage. This year we improved 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 the one from 19BNU, since we changed the temperature sensing riboswitch from RNA thermometer FourU to NoChill-06.
Due to the impact of the pandemic, we have not been able to conduct the confirmatory experiment, but we provide an experimental plan. You can find this on our Experiments page.
At the same time, we used the original data from 19BNU for modeling verification. More detailed information is showed on our Modeling page. Modeling proves that the kill switch is theoretically reliable and can be experimentally verified. You can also read the Engineering page for more details.
Current available of Kill Switches
We obtained valuable information based on 16Marlborough Team's Kill Switch database. We traversed mostly used 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 using 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 by Cas3 | ++++ | Note 1 |
Note 1: The method of can gene knock out by 2019 Grand Prize winner is ingenious and effective. Unfortunately, it cannot be applied to our project. It has been 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 (fever). Moreover, it requires to express two types of proteins which imposes potential 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 PT-αCRP initiates 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 | + | -- | BBa_K3096046 | Metabolites as responding factors, are greatly affected by food intake. | |
| Other conditions | N-Acetyl-Glucosamin-6 phosphate regulated repression system | + | -- | BBa_K3096048 | It is not very efficient. |
| CO2 concentration | ++ | ---- | can gene knock out | Note 1 | |
| Light condition | + | - | killer red | 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.
This contribution is mainly made by our team member Jingqi. You can enter our Team page to learn more about her.
Basic parts
Osteoporosis and other diseases caused by a calcium deficiency are common problems among the elderly in many regions such as China and India. We have discovered CaAP (Calcium Absorption Peptides), a class of oligopeptides that can chelate calcium and promote Caco-2 cells to uptake calcium, which can alleviate people's calcium deficiency problem, especially the elderly. This will provide help for the health care of the elderly in our area and is of good social value.
We learned from literature and designed the entire CaAP expression peptides sequence contains five different calcium-binding peptides, all of which have been proved through experiments to significantly increase calcium absorption in vivo or in vitro. Calcium ions spontaneously bind to the peptide's Asp or Glu residue, and calcium influx into intestinal epithelial cells is increased. The oligopeptide sequence is expressed as a complete peptide in the engineered bacteria and is cleaved into functional calcium absorption peptides in the presence of digestive enzymes in the intestinal lumen.
We have provided a new part CaAP to the Registry. We obtained new information from the literature to support our project and provide new experimental data on the expression of CaAP in E. coli for future teams who will use the parts. You can read our Parts page for specific information.
This contribution is mainly made by our team members Zhang Zhenru and Lu Yiyang. You can enter our Team page to learn more about them.
Composite parts
We have noticed that the treatment of calcium deficiency is mainly aimed at the elderly. If we choose the treatment plan that patients take CaAP products regularly, they will likely forget to take the pills, and the efficacy will probably be unsatisfactory.
We learned from 19Fudan Team, who had tried to make lactase controllable expressed in the intestine to treat lactose intolerance. We decided to establish a safe and controllable CaAP expression system using quorum sensing. The special promoter luxPR regulates the expression of CaAP. Under the regulation of our quorum-sensing system, bacteria's proliferation will be inhibited when they achieve the appropriate population, and CaAP expression begins. You can read our Parts page for detailed information.
This idea allows the engineered strains to act directly inside the human body, which is an effective way to replace the target substance synthesized in vitro and administered regularly. More importantly, it considers the elderly's difficulty. The commercialization of our project woould result in a market transformation and has efficient social significance.
We improved the existing 19Fudan quorum sensing system. We increased the secretion of antimicrobial peptides by adopting a relatively low expression of antimicrobial peptides (mcbABCD) and high expression of the antimicrobial peptide immune portion (mcbEFG) from the reversed DNA strand. Those changes made our quorum system much more efficient and sensitive.
This contribution was made by our team members Li Mingwei and Zhang Gaochen. You can go to our Team page to learn more about them.
Improved part
The part above has met Golden Medal criteria #2, which means we changed the sequences, and it improved an existing Part's function. We also did experiments with both the improved part and the original part as a control.
Parts collection
At the beginning of the experiment, we found that the circuit we designed could not operate successfully. We set out to construct a series of plasmids containing different parts for troubleshooting, hoping to assemble a complete circuit after removing the problems.
Our trouble shooting process provides a practical reference idea for any team who wants to study bacterial secreted proteins in the future. When they find that the complete circuit cannot work effectively, they can conduct experiments on individual parts separately, eliminate the fault, and finally re-assemble.
In result, we have provided a set of plasmids that these teams can use to solve their problems, which shows we are willing to put the idea of engineering and knowledge sharing into practice. You can read our Parts Collection for detailed information.
This contribution was made by our team members Liu Yuanyuan, Lu Yiyang, Zhang Zhenru, Li Mingwei, and Zhang Gaochen. You can go our Team pageto learn more about our team members.
Measurement
Measure trace calcium on a test strip
Insufficient calcium intake or excessive calcium supplementation can cause a series of physical symptoms among the elderly. Therefore, it is necessary to provide convenient blood calcium testing for them. Routine blood calcium testing is cumbersome, so we try to improve the blood calcium detection method and provide the elderly with a simpler one that reduces drown blood volume. We hope they can conduct daily self-monitoring of their blood calcium status.
We have learned that the principle of measuring blood glucose through test strips. Glucose will generate red quinones with the effect of enzymes, which causes a change of absorbance. We can measure glucose concentration by detecting the intensity of light reflected by the test strips. The calcium ion in the serum can combine with methyl thymol blue and form a blue compound in an alkaline solution, which can also be determined by spectrophotometry. Therefore, we consider making test strips for measuring serum calcium ion concentration following the blood glucose measurement method. You can read our Measurement page for more information.
Hardware
We also designed a supporting portable blood calcium meter with the simplest kinds of stuff, low-cost LED tubes and an existing mobile phone app. And we consider integrating blood calcium detection functions into existing blood glucose monitors. This kind of hardware has a simple structure and convenient operation, suitable for the elderly. Blood calcium can be detected with a small amount of blood sampling drown from fingers with a high detection sensitivity.
Our testing method helps discover the problem of excessive calcium absorption caused by probiotic activity, which can be easily solved by taking arabinose tablets in pharmacies (please read to our Safety page). It also provides users with reference data records of body calcium status. This is not only simple and clear in theory but also feasible in practical applications. We hope that the commercialization of our design can increase the attention to the elderly's health and promote social justice.
This contribution was made by our team member Ziyuan. You can go to our Team page to learn more about him.
What we have done for the iGEM community:
- Education: Popular biological science courses for the elderly
- Human practice and entrepreneurship: A complete business plan for transformation of scientific research results into commercial products
- Modeling: Improved modelings of the existing team to make it suitable for our project
- Kill switch: A more sensitive kill switch, ideal for the human body
- Basic part and Composite part: A kind of calcium absorbing peptides expressed in E. coli and excreted in the human intestine
- Parts collection: A series of plasmids for troubleshooting
- Hardware: A new method to detect blood calcium and a hardware intended for the elderly
Signature: Tianhui