HUMAN PRACTICE

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Project Determination

At first, we were interested in decreasing the price of cordycepin, for we thought a lower price could contribute to a more popular usage and more scientific studies. However, we needed to know the methods of producing cordycepin on the market now and their advantages and weaknesses, so that we would know how to improve. Then we did a research and found that the methods of producing cordycepin on the market mainly include biosynthesis and chemical synthesis. We compared these two methods and realized biological synthesis has a greater market value, so we decided to choose the biosynthesis route to make further improvements.

In CCiC, the judges said that we needed to extrude our highlight, which could be listed as the potential value of biosynthesis cordycepin, the cost comparison of traditional way (chemical synthesis) and biosynthesis way to produce cordycepin. This suggestion reflected that our research on the comparison was practical and meaningful.

Then we had a consultation with Professor Huang Jing. Professor Huang Jing is a specialist in the field of natural medicine and he is also a good example combining scientific research and industrialization that of great value for reference. He told us that the traditional method to produce COR isn't satisfactory, our working has not only a potential market but also advantages in industrial production, which helped us to make our minds to do this project.

Then we went to Purify, a professional compounds-purifying (Cordycepin is one of their main target products) company in Chengdu for a field trip. Mr. Qiling Xie from Purify told us that the yield is the golden rule in the industry and we don’t need to worry about the technical problems in the purification of cordycepin for it is already mature. Thus, we decided to focus on increasing the yield. This helped us build a much more industry-friendly project.

Improve Project

After determining the project, we found that there were many problems that remained to solve. Therefore, we got connected with the discoverer of cordycepin in Cordyceps millitaris, professor Wang Chengshu in the Shanghai Institute of Plant Physiology and Ecology. We got two important messages: the function of NK domain of Cns3 is unclear, and the expression of Cns3 in yeast is unclear. So we decided to study more about hisG domain of Cns3 instead of NK. Another reason for giving up NK domain is that the product catalyzed by NK domain can be got from RNA degraded.

In CCiC, the judge mentioned we could build a library of the linker between cns1 and cns2 protein. So we searched the relevant papers and found some regularly-used linkers. Then we used modeling to go through all the linkers that we had found and conducted preliminary screening.

Experiments

During the following experiments, we encountered many problems as well. Luckily, most of them were solved with our toil and others’ assistance.

We needed a signal to represent our protein, and Peking generously gave us the yeGFP, which can express the protein optimized for yeast.

As we all know, the iGEM team of Tianjin University, as a senior yeast team, has used yeast as a chassis for many years and has rich experience in yeast transformation and culture. During the CCiC in 2019, we exchanged information on the electroporation and plasmid extraction of Saccharomyces cerevisiae with Tianjin University. The members of Tianjin University provided us with many suggestions. Based on the recommendations of Tianjin team, we have revised our experimental procedures and the results have indeed improved to some extent.

At first, we used Saccharomyces cerevisae AH109 as our chassis to construct Met/Gal4 delay expression system initially. But we couldn’t get desirable monocolony in SD-Ura plate. Considering professor Liu Ke is a specialist of yeast, we consulted him for some guidance. After talking with Professor Liu, we knew that AH109 contains Ura gene. This gene stopped us from screening yeast in SD-Ura plate and made yeast congregated. He advised us to choose other kinds of yeast for screening in SD-Ura plate. Thus, we referred to the internet for information and finally determined to use S.cerevisae YM4271.

Professor Cao Yang helped us with modeling. In the situation of being blocked by the poor structure prediction result of the enzymes by homology modeling, we turned to Prof. Cao Yang for help. He listed the weakness of the utility of homology modeling, relying heavily on the coverage and quality of the template structure. He recommended another type of structure prediction methods, called fold recognition, which has another alias, protein threading. This method is more suitable in our case as we can not find proper templates.

When we conducted our molecular kinetic simulation, Prof. Cao gave us support for the Linux environment and the software package, GROMACS, running on it.

Commercialization

To make sure that our product does have practical value and can adapt to the market requirements, we conducted a questionnaire and made a business plan to help our product to be commercialized.

We had a consultation with professor Lili Wang from the business school of Sichuan University. After we introduced our project, professor Wang suggested that we conduct market research to determine product positioning. So, we conducted a questionnaire to discover the potential markets and consumer groups of biosynthetic cordycepin.

In China iGEM Southwest Meeting, iGEM Asian Ambassador, Nan Zhang’s lecture and advice made us reexamine our project. We designed and analyzed our questionnaire survey scientifically. Meanwhile, we also investigated the industry’s background and designed a business plan for the full commercial value of our product.

To ensure our engineering yeast has a market prospect and can become an industrial product, we made a business plan. In the business plan, we have referenced the result of our questionnaire: (1) We’d better publicize our biosynthetic cordycepin combined with traditional Chinese medicine (TCM) via new media. (2) Small profits but quick turn over are thought to be a better way to expand the market. (3) Young people who know transgene are more willing to accept biosynthetic cordycepin. What’s more, in the meet-up with SCU-China, NWU-China, and YAU-China, they suggested that it could be better if we compared the existed produced method with ours in more detail. So we added more comparisons in the business plan which can highlight our strengths.