Contributions in modeling, circuit design and human practice
Our team has made contributions to the iGEM community in all aspects. For modeling, we improved the mathematical model of the existing team to make it suitable for our project. For safety, we summarized the existing kill switches' information and improved our kill switch, making it more sensitive and ideal for the human body. For basic parts and composite parts, we found calcium absorption peptides from literature, which help intake calcium. We hoped to make it expressed in E.coli and excreted in the human intestine. For parts collection, we built a series of plasmids for troubleshooting and provide them for future teams interested in secreted proteins. For measurements, We found a new method to detect blood calcium and tried to design supporting hardware intended for the elderly. For human practice, we provided popular biology science courses for the general public, promoting educational equity.
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 2019-Fudan Team and use the random process to describe it more clearly to model the quorum sensing process in stages and levels.
Kill switch
In the kill switch section, we use the 19-BNU team's data, improve the logistic equation, adjust the promoter properties, and make 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 the Team Page to learn more about team members.
Kill switch
New information from literature
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.
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
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 2019 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 19BNU-China, 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 Notebook. At the same time, we used the original data from 19BNU-China for modeling verification. More specific information is showed on the Modeling Page. These above proves that the kill switch is theoretically reliable and can be experimentally verified. You can read the Safety Page for more details.
A summary of kill switches
We also shared more valuable information based on the 2016 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.
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 Zhang Jingqi. You can enter the Team Page to learn more about our team members.
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 that 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 CAAPs in the presence of digestive enzymes in the intestinal lumen.
We have provided a new part CAAP genes for the element library. 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 the Experiment Page for specific information.
This contribution is mainly made by our team members Lu Yijun and Zhang Zhenru. You can enter the Team Page to learn more about our team members.
Measurement
A new measurement method
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-detection 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.
hardware
We also hope to design a supporting portable blood calcium meter with the simplest kinds of stuff like LED lamps and mobile phones, or integrate 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(refer to the 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 the hardware can increase the attention to the elderly's health and promote social justice.
This contribution was made by our team member Wei Ziyuan. You can go to the team page to learn more about team members.
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 the 2019 Fudan 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 based on the prokaryotic Escherichia coli. The special promoter luxpR regulates the expression of CAAP. Under the regulation of the quorum-sensing system, the bacteria's proliferation will be inhibited when they achieve the appropriate population, and the CAAP expression begins simultaneously. You can read the composite parts page for specific information.
Suppose it can be successfully colonized in the digestive system so that the flora can secrete CAAP regularly and quantitatively to promote calcium absorption. In that case, it will provide a more feasible solution to the lack of calcium absorption.
This idea allows the secreted strains to act directly inside the human body, which is an effective way to replace the target substance synthesized in vitro and administered regularly. This plan provides an innovative solution for future teams. More importantly, it considers the elderly's difficulty. It is conducive to the commercialization of project results in the market and has efficient social significance.
This contribution was made by our team members Lu Yijun, Zhang Zhenru, Li Mingwei, and Zhang Gaochen. You can go to the team page to learn more about the team members.
Parts collection
At the beginning of the experiment, we found that the circuit we designed could not operate successfully, so we set out to construct a series of plasmids containing different parts for troubleshooting, hoping to assemble a complete circuit after removing the problems. The solution 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 reconnect.
Remarkably, we have provided a set of plasmids that these teams can use to solve their problems. You can read the Parts Collection Page for relevant information.
This contribution was made by our team members Lu Yijun, Zhang Zhenru, Li Mingwei, and Zhang Gaochen. You can go to the team page to learn more about the team members.
Human practice
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