We interviewed two researchers, Mr. Bai and Ms. Li from Shenzhen Research Institute, Tsinghua University, to give us a more comprehensive understanding of today's testing solutions and give some advice on our method design.
It really matters and then like it really doesn’t matter. What matters is the people who are sparked by it. And the people who are like offended by it, it doesn’t matter.
As Mr. Bai suggested, despite many developed methods in the market, there were still areas for potential improvements, such as its safety, efficiency, and convenience. He emphasized convenience in particular since most testing trials require the operation of heavy, expensive, and immobile machines. He suggested that we could invent a small, portable and cheap device. Following his suggestion, we researched and referred to the past iGEM teams. We were inspired by the 2017 iGEM team TUDelft and designed our first draft of the hand-powered centrifuge.
Mr. Li pointed out a crucial flaw in the current testing methods - the "quantitative testing" of the process is difficult to eliminate all errors when testing for multiple pollutants due to a margin of error and the complexity of the operation.
The type of material we use to build our device is an important factor in deciding its cost of production and hence its price level. However, this is not a problem for us: we used biodegradable plastic for our testing kit packaging, remaining our system low-cost and highly accessible for scientists.
Initially, we planned to link the two gene modules of lysis and chromogenic directly after a susceptibility promoter. It was envisaged that the two modules could be initiated together under the induction of one promoter to achieve our final detection purpose. In our first version of the design, we used two inducible promoters – the pBad/araC promoter (BBa_10500), the arabinose promoter, and the copper-sensitive promoter (BBa_1760005) to activate our cleavage and The expression of the chromogenic module, and, we designed a relatively strong RBS (BBa_B0034) inserted after the promoter, and inserted the RBS again after the ß-galactosidase gene, and then inserted the SRRz cleavage gene. At the end of our entire gene pathway, we use a double terminator BBa_B0015 to stop transcription, a multiple-use and powerful terminator that can effectively terminate gene transcription.
In our first verification experiment, we found that even if the color changed successfully, meaning that our promoter started the translation of the ß-galactosidase and SRRz lysis genes as we hoped after receiving the signal factor, ß Galactosidase successfully performed a chromogenic reaction with x-gal. However, we found that the chromogenic reaction was not as expected. Because the bacteria failed to work, meaning they decomposed and died before sufficient ß-galactosidase was produced, which caused our entire detection system to fail to meet the standard. Therefore, we need another genetic pathway to ensure that the bacteria can remain active until sufficient amounts of ß-galactosidase are produced. With this purpose in mind, we interviewed Mr. Bai and Mr. Li, researchers conducting synthetic biology research at Tsinghua Research Institute in Shenzhen.
After Mr. Bai understood our problem, he suggested that our deficiency in gene pathways is reflected in using one promoter to induce two gene modules. Mr. Bai believes that in our initial design, the two gene modules are simultaneously controlled by the susceptibility promoter as a whole, so the lack of independence between the two gene modules will lead to, as we mentioned above, the lysis module has already started to run and terminate the transcription of the chromogenic module before bacteria were able to complete the transcription., resulting in the production of insufficient results. We took in Mr. Bai's suggestion and decided to treat the chromogenic module and the lysis module independently to ensure that both modules could work to their full potential.
In order to ensure the accuracy and stability of gene pathway expression, we interviewed another researcher, Mr. Li, from the Research Institute of Tsinghua University in Shenzhen, on selecting the chromogenic module's promoter. After communication, he believes the chromogenic module's promoter needs to be accurate and potent. He suggested that we use the T7 promoter for the chromogenic module. The T7 promoter is a strong promoter derived from T7 phage and can specifically respond to T7 RNA polymerase. Since it only transcribes the DNA sequence downstream of the T7 promoter, it guarantees high accuracy and powerful work efficiency. In addition, following the suggestions, we inserted a BBa_B0015 double terminator (a strong terminator that can effectively terminate the operation of the T7 promoter) at the end of the chromogenic module to ensure the smooth expression of the subsequent lysis module pathway.
With the help of the above two teachers, we inserted an RBS (still BBa_B0034) before ß-galactosidase’s reaction with the promoter and inserted a T7 promoter before the RBS promoter to initiate the expression of the chromogenic module gene. In addition, a BBa_B0015 double terminator was inserted downstream of the ß-galactosidase gene to terminate transcription of the chromogenic module. Finally, it was found that the color expression of the modified detection system is more accurate and distinct, greatly improving the reliability and accuracy of our detection system.See more in our Proof of Concept Page and Engineering Page.
Meeting
We held a roundtable discussion with other teams about safety in order to improve our biosafety. We each shared information about safety issues that we had identified during previously completed experiments. In our conversation with LZU-HS-Pro-B, we discovered that we had not previously considered partitioning the lab to avoid the mixing of drugs that could lead to hazards, and we have since improved this issue. In addition, one of the teams attending the meeting also mentioned that they had chosen biodegradable materials for their product packaging, and we were inspired to make changes to our hardware.
In the first EPIC meeting, we learned about project management issues and the strategic layout of the project through Mr. Lu Lingyun. After this meeting, we found out the problem of duplication of tasks and improper utilization of staff in the previous teamwork. In order to work out the project more efficiently in the future, we referred to the "specific tasks, multiple teams" system mentioned by Mr. Lu in the meeting to allocate tasks, subdividing the tasks under the three team leaders, and then set up an independent person in charge for management. In addition, in terms of the future business planning of the project, we decided to develop our business in the future, thanks to the "horizontal development of the project" mentioned by the teacher in the meeting, and considering the extensibility of our project - we can use similar methods to test multiple substances. Multi-product line development.
During the second EPIC meeting, Mr. Xiao shared with us that one of the major risks that they would consider as investors are the policy risk. We thought that the government is now raising the standard requirements and regional restrictions on factory emissions and the factory location, and if we can't settle this anxiety for investors in our business planning, it may lead to mistakes in the future financing process. Therefore, during the Q&A session, we asked the instructor about the factory requirements for the bioassay manufacturing industry. Through Mr. Xiao's answers, we were able to refine biosafety further in our future plant. Firstly, we will set up separate functional rooms in our future plant to avoid the risk of the crossover of biochemicals. Secondly, we will conduct further research on raw materials and set up strict standards for air flow rate, temperature, and moderation in the room to ensure the safety of reagents and drugs in the plant.
We also learned from communicating with Mr. Li Huaiyu that as we can assay different substances by simply changing the promoter, each customer's requirements may be different, and direct batch production may cause our products to lag in sales. He, therefore, recommends that we should first meet a certain number of orders before producing reagents for the detection of a certain substance. To do this, we can seek direct cooperation with the companies involved to ensure a continuous order quantity and to allow each production line to reach maximum utilization.
Visit
Marketing strategies and financial planning are crucial for our testing kit, considering our future plans of standardization and batch production of this product. Therefore, we sought direct cooperation with several successful businesses and potential investors. In order to develop our business model, we visited Shenzhen Cell Bank to seek some insightful advice on utilizing our production line and product marketing strategies.
Shenzhen Beike Biotechnology, the owner of Shenzhen Cell Bank, was recognized by the College of American Pathologists for five consecutive years. Beike Bio has passed the CAP quality assessment in a number of projects, including testing of clinical trials, and has achieved international standards in the standardization and accuracy of testing, information analysis, and clinical interpretation of the validation projects. Being an outstanding company in standardizing testing trials, their guidance on consumer behavior analysis, advice for financial planning, and strategies on factory location and production line helped to solidify our marketing map.
During the CCiC event, through sharing and exchanging projects with other Chinese teams, we reviewed the shortcomings of our previous process. At the very beginning, in terms of education, through our own presentation, other teams participating in the event questioned us: we had not considered the target audience, and the content of the educational activities was slightly professional for the general public, which might not lead to a good educational effect in the end. In addition, through the feedback from the judges on the spot, we learned that the explanation of the experiment in our presentation was not clear enough, such as in the PowerPoint should be more through the flow chart to explain. Finally, in the Human Practice section, we did not investigate our target customer, the testing laboratories and NPOs, in detail.
After this, we again conducted an orientation education for middle school and high school students, and at the same time, in order for them to better absorb the content of our lectures, we made pamphlets to distribute in advance so that our audience could have some understanding of the content of our lectures before our presentations. In addition, we re-visualized the experimental process to give the audience a clear understanding of our project. After all, we interviewed some managers in NPOs and experts in detection labs.
To reinforce our existing knowledge of probiotics and to learn some possible extensions of our detection system, our advisors contacted Mr. Si from Zhongke-Jiayi Bioengineering to hold a lecture about probiotics and their impacts on the human body. In the lecture, Mr. Si talked about some newly emerged areas of research, some future directions of probiotic research, and its results. We gained some helpful insights on our potential areas of development, which is the possibility for our product to detect organic compounds in samples from patients. For the record, we strictly follow the safety rules of human body sampling. All of the samples will be highly standardized, commercialized, and monitored, and the samples taken out will be disposed of in a secure. In the future, our product could be developed to detect calcium ion concentration in blood, blood glucose concentration, and urine concentration, all of which could be implemented in clinical trials. Therefore, our potential stakeholders were no longer limited to testing institutions and related research, but also areas of medical practices.
