In the process of many iGEM teams carrying out projects, it is often impossible to develop projects that can be applied to life given the cost and schedule. In order to avoid this situation, we draw on the waterfall model in software engineering as a development model, which is divided into feasibility analysis, requirements analysis, project design and project realization. Human practices is an indispensable tool for us throughout our entire process. We also hope that more iGEM teams can use such models to develop projects.
In the course of our brainstorming, some students proposed the idea of degrading antibiotics. After having the idea, the first step is the feasibility analysis. If we don't think about whether we can do it, we will blindly carry out the project, then our project will ultimately lack practical significance. In order to determine whether the project is feasible, we organized and participated in the 5th Southwest China iGEM Exchange Conference to determine the technical feasibility and visited the fourth water purification plant in Chengdu to determine the application feasibility and discussed the feasibility of policy. Through this series of activities, we decided to pay more attention to expired drugs.
In our brainstorming process we have proposed the idea of degrading ciprofloxacin. In order to determine the technical feasibility, we held the 5th Southwestern iGEM Exchange Conference with UESTC-Software at the University of Electronic Science and Technology of China. We invited Ms. Zhang Nan, Ambassador of Asia, to explain the concept of human practices to us, and invited BM-AMU, SCU-China, SICAU-iGEM, XJTU-China and SCU-China to exchange ideas.
“During the process of project demonstration, students from SCU-China and Ms. Zhang Nan made suggestions: Pay great attention to safety issues, because if genetic escape occurs, it will cause great impact on the environment.
So according to their suggestions, we designed the kill switch and decided to use the semantic containment methods to further control our E.coli.
After we had the idea of degrading antibiotics, in order to determine the feasibility of the application scenario, we went to the fourth water purification plant in Chengdu to understand the source of antibiotic pollution.
We learned that the dirt is first filtered through the pretreatment system and the membrane grid station. In the MP-MBR system, activated sludge is used, and microorganisms in the activated sludge are used for degradation, and the MP-MBR membrane system filters out pollutants larger than 0.1 μm and most bacteria. The UV is then sterilized again to prevent damage to the machine that actually runs the diaphragm.
“The technicians told us that the main sources of antibiotics in the sewage flew into sewage plants are veterinary antibiotics used in cultivation and medical antibiotics used in pharmaceutical plants, hospitals and families.
We decided to learn more about how to treat these three sources of antibiotics.
In our subsequent investigations, it was found that antibiotic pollution of expired drugs pollution has not received widespread attention. Among the 663 cities in China, most cities treat expired drugs as domestic waste. Shanghai has relatively perfect recycling system of expired drugs, and expired drugs in Shanghai are classified as hazardous waste. The main treatment process is the rotary kiln incineration process which has a variety of pollution pathways. So in the process of our local government collecting the draft garbage classification, we proposed to the local government to set up a specific point of recycling drugs.
“We found that 99% of cities in China have a lot of room to improve regulations on the treatment of family expired drugs.
We recommend that the government should set up some specific expired drugs collection points.
Combined with the above investigations and analysis, from the aspects of the technique, application, policy, we want to make a project to recycle and degrade expired drug which is closest to our lives.
Demand analysis has a major impact on the entire project development phase and is the basis for project development. Insufficient demand analysis can lead to a series of problems, such as cost and budget out of control, project design can not meet users' needs. In order to accurately analyze user needs, we distributed more than 200 questionnaires for analysis which helps us decide to design the expired drugs recycling bin, drugs avenger, and visited a number of chain pharmacies to determine the function and performance of the recycling bin.
User needs analysis
In order to determine whether people have special requests for recycling device, we have released a questionnaire on the status of expired drugs, and a total of 240 valid questionnaires were collected.
“ 78.33% of respondents confirmed or intend to think that there were expired drugs in their homes. It can be seen that the phenomenon of expired drugs in the family is still very common. What's more, most respondents didn't pay enough attention to their use of drugs and prefer nearer recycling position. People prefer to have an expired drug recycling dusbin near their home.
So we decided to make an expired drug recycling and degrading bin, and use mathematical modeling to determine where the bins are placed.
Moreover, according to our survey, the elderly took the most expired drugs. These allow us to decide to regard elderly people as the project audience and pay attention to families with elderly people, improve the living habits and knowledge level of the elderly, and verify the practicality. In order to be more helpful to the elderly, we have also carried out educational activities on the elderly.
In order to determine whether our recycling bin requires other functions, we visited several pharmacies, introduce our project to the store manager, pharmacist, salesperson and other staff around the treatment of expired drugs and sought their advice. They questioned our bins, is it safe to use E.coli degradation chambers? Will it do harm to people's bodies? Where will this recycling bin be placed? Is it possible to give some rewards to encourage people to throw expired drugs into the recycling bin?
We need to be more cautious about security issues. In addition to kill switch and semantic containment, we use UV sterilization technology in the recycling bin. We need to use the mathematical modeling of the recycling bins to determine how our recycling bins are set up in the city. We also think about what can be used as rewards to encourage people to use our projects.
In the process of project design, using modular design principles, it is divided into three modules: synthetic biology design, hardware process design, and device layout design. In this process, we exchange ideas with five teachers and constantly improve our project.
Synthetic biology design
In order to quickly produce a large number of degrading enzymes after antibiotic is detected, our PI suggests us to design quorum sensing system.
In order to increase the degradation rate, we have designed quorum sensing system.
The upper bacteria produced AHL after detecting ciprofloxacin, then AHL combined with LuxR which is often expressed by the underlying bacteria to induce the expression of CrpP (degrading enzyme).”
Hardware process design
We planned to use microfluidic technology to encapsulate E.coli with alginate solution, CaCl2 solution and water. The gel beads formed can allow certain enzymes produced by E.coli to be released to the outside world, while E.coli can not be produced. Microorganisms in the environment also do not enter the gel. And E.coli can live for several days. However, we still have doubts about hardware fabrication, microfluidic chip fabrication, embedding and cross-linking methods, gene influx release, and E.coli survival. Just as we thought about the harmful substances entering the microbeads, the risk of cell escape, and so on, it coincided with Professor Yin Huabing from the University of Glasgow's biomedical engineering program, and we asked her about these problems.
“1. In terms of light source made by hardware, Professor Yin believes that it is feasible to use laser, but the teacher recommends using a light source: X-Cite LED fluorescence microscope to excite the light source.
However, due to funding constraints, we decided to use laser light sources after repeated consideration.
2. The design of microfluidic chips is the most important and the most difficult step. The design process is too specialized, and the microfluidic chips currently applied to cell encapsulation are less, and the technology is less mature. Even if the chip is designed, it may take dozens of adjustments to achieve the function, and the cost is too high.
Taking the comprehensive consideration into account , we decided to abandon the use of microfluidic technology.
Device layout design
In order to make the processing device in the project can be placed reasonably in the community, we apply mathematical methods to solve this problem. The device layout in the community properly depends on the structural characteristics of the community, the population of the community and the longest distance to walk residents can accept, etc. The lack of consideration of these factors will cause the irrational position of the device, so we have established a mathematical model to scientifically place the device.
After we made the second-generation device, we used the data detected by it to analyze the resolution of the acquisition card together with the teacher, and finally selected the appropriate 24-bit data acquisition development board.
The teacher also provided suggestions for the design of the device and the circuit design to solve the problem of centralized power supply for pumps, lasers, development boards and other equipment.
Professor Ke Chen taught us about the type and working principle of the detector, analyzed the advantages and disadvantages of common photoresistor, avalanche diode and photomultiplier tube in terms of cost, function, size and difficulty of use, and discussed the difficulty and cost of the self-made filter and amplifier circuit.
Finally, the plan to purchase an avalanche detector with its own amplifier circuit was decided.
In order to complete our hardware, we learned the basic operation and precautions of 3D printing from Professor Xiangyu Mao. With the help of Mr.Mao, we analyzed the advantages and disadvantages of fused deposition modeling(FDM) in rapid prototyping and photocuring. After taking these advantages and disadvantages into account, we finally decided to use ABS for photocuring 3D printing.
“Considering the accuracy and cost of 3D printing,
we improved the optical path and detail design of the device
, and used 3D printers in Mao’s laboratory to print 3D printing of the second generation device to verify the design flaws of device in actual use. It greatly helped us to upgrade the device.”
Bacterial box design
“Dr. Gul provided suggestions for the design of our cartridges.According to our needs,
he proposed that we should make the interior of the capsule in the shape of funnel and he also pointed out the design requirements for the shape of some important details. We applied his suggestions to the design of our third generation device.