Diabetes mellitus is one of the most important chronic diseases that reduce the quality of human life. China's population is estimated to be about 1.4 billion, and the world's total population is estimated to exceed 8 billion. The number of diabetic patients in China exceeds 140 million, and the number of diabetic patients worldwide is estimated to be about 500 million.

Drug treatment is one of the main means of diabetes treatment. According to the Blue Book of the Oral Insulin Industry, the size of China's diabetes market is expected to reach 75.1 billion yuan in 2023, an increase of more than 20% from 62.4 billion yuan in 2022. By 2030, the market size is expected to reach 132.3 billion yuan.

In recent years, with the rapid development of diabetes drug therapy, a variety of new hypoglycemic drugs have entered the clinical practice. By reading the BMJ published in SCI Q1 on April 6,2023, Impact factor of 93.6 Benefits and harms of drug treatment for type 2 diabetes: systematic review and network meta-analysis of randomised controlled trials and other literature to understand the strengths and disadvantages of commercially available drugs, We found two problems with the currently available drugs that are poor efficacy and high side effects:

Poor efficacy: The results of the above literature show that only SGLT-2 inhibitors and GLP-1 receptor agonists were reduced in the core indicator of all-cause death. Non-steroid mineralocorticoid receptor antagonists and metformin may reduce mortality, and other drugs may have little or no effect or uncertain impact on mortality outcome. The hypoglycemic drugs on the market are disappointing.

Side effects: In the first half of 2023, the fire drug GLP-1, the market share of the Chinese market, reached a sales of 3.4 billion yuan, but the GLP-1 receptor agonists (1.97,1.39 to 2.80) probably increase the risk of severe gastrointestinal adverse events.(GLP-1 receptor agonists (1.97,1.39 to 2.80) may increase the risk of serious gastrointestinal adverse events) with their side effects.

So we turned our eyes to the new drug —— hypoglycemic peptide P37, which is undergoing clinical trials. We read the Native polypeptide vglycin prevents nonalcoholic fatty liver disease in mice by activating the AMPK pathway literature and found that P37 showed a comparable therapeutic effect to metformin in a model of type 2 diabetes. In a clinical experiment of 170 cases, P37 formula significantly reduced fasting and postprandial blood glucose levels in patients with type II diabetes, with a response rate of 83%. And has the advantage of medicine / food homology, to eliminate the side effects from the source, is the ideal hypoglycemic drugs in the future.

Our team in consulting the drug research and development director professor yao morning when we found that hypoglycemic peptide P37 current production method is comparing the original from soybean with physical and chemical method to extract, extraction efficiency is low, raw materials for important food crops such as pain points, serious limit the drug capacity, and so large-scale, low efficiency of the use of food, against the interests of all mankind and ethics. So we asked yao the morning professor team, we want to use the method of synthetic biology, with the yeast to synthetic hypoglycemic peptide P37, to solve the problem of raw materials for food and low capacity, professor yao morning greatly appreciated and provide the support of our team experimental conditions and as our first IP instructor, our topic out to synthetic hypoglycemic peptide P37 thus established.

In order to understand the current domestic market of hypoglycemic drugs and the expectation of diabetic patients for hypoglycemic drugs, we came to the activity center of the retired staff to conduct a voluntary blood sugar survey for the retired staff. In total, we measured blood glucose in 100 middle-aged and elderly people, 6040 men, 24% 40 to 60 years, 21% 60 to 70 years, 38% 70 to 80 years, and 17% 80 to 100 years. According to the official blood sugar concentration, we divided the blood sugar status into five grades: excellent, good, general, poor and extremely poor. 43% of the elderly had excellent blood sugar, 26% had good blood sugar, 14% were poor, 13% and 4% had poor blood sugar.

20% of them had a history of diabetes, but only 60% of them chose to use hypoglycemic drugs, and the remaining 40% refused to take or stop taking hypoglycemic drugs. According to the MMAS-8 medication compliance scale, it is concluded that most of the hypoglycemic drugs on the market have low medication compliance problems.

In our further investigation of the causes of low medication compliance, we found that only 33.3% of the elderly recovered above normal blood sugar after taking hypoglycemic drugs, and many hypoglycemic drugs did not have obvious hypoglycemic effect. Among the elderly who refused to take or stopped taking hypoglycemic drugs, 40% said that they have basic diseases such as cardiovascular, liver and kidney, and there are no hypoglycemic drugs suitable for them on the market or fear of the side effects of hypoglycemic drugs on the market. Thirty percent of the elderly said that hypoglycemic drugs are too expensive to take as a treatment for chronic diseases. The remaining 30% of the elderly did not realize the harm of long-term high blood sugar and chose to ignore it.

Therefore, we conclude that the reason for the low compliance of most hypoglycemic drugs on the market is that they have insufficient obvious effect and high side effects, which is not suitable for the elderly with basic diseases, expensive, most of them use injection, and the operation is difficult.

Drug compliance is low and leads to poor results, with 20% of older people who have a history of diabetes or refuse to take diabetes having poor blood glucose and 40% being extremely poor.

After we presented our practice data to Professor Li Hanluo, in the discussion with Professor Li Hanluo, Professor Li Hanluo said: the market is in urgent need of an oral, polypeptide or protein, low toxicity, low cost, medicine / food homology drug.

At present, P37 just meets all the above requirements, and our team uses synthetic biology and synthetic biology to reduce glycopeptide p37, which can further increase the yield and reduce the cost.

At the same time, our team realized that to raise the public awareness of diabetes, offline publicity is far from enough, which needs to rely on online media. Therefore, our team later focused on we-media (hyperlink).

In addition, our team members also visited BGI and listened to BGI's treatment plan for chronic diseases is to treat diabetes by adjusting the nutritional structure of food and adding the proportion of vitamin b. We also reported our work to BGI. One of our advantages, the homology of medicine / food, coincides with BGI's plan, which was highly praised by Expert Liu Chen of BGI.

After collecting all the background of the project, we started to prepare the preparation of the project. The hypoglycemic peptide P37 is a polypeptide, the expression product, and our team used the central rule to reverse the possible base order of all genes that can encode such polypeptide. Finally using our algorithm, in order to get the highest gene stability, the least easily in the experiment, the production variation, avoid restriction sites, avoid in the middle of the gene as the basic principle, design and synthesis of target gene (vglycine, after all referred to as VG), detailed work reference model part (hyperlink).

Through communication with our tutor, Professor Yao Chenguang, and analysis of objective experimental conditions in the laboratory, we set the collection method of hypoglycemic peptide P37 as by nickel column adsorption and eluted by imidazole competitive ligand, so we added histidine (his) label at the 3 ′ end. DDDK enterokinase was added to the site where the antidiabetic peptide P37 was separated from His. Therefore, our initial gene design was pPIC9K-3His-DDDk-VG.

In terms of the selection of engineered bacteria, our team referred to the 2022 SCUT-China's clever use of Pichia to produce two natural terpenoids, demonstrating the great potential of Pichia. Later, through our team held a meeting to discuss, we found that Pichia is very suitable as our drug hypoglycemic peptide P37, a small molecular weight polypeptide. Our team summarized the three advantages of Pichia: First, because it enables proteins to fold correctly, the protein expressed has a high biological activity. Second, the expression time of the protein can be regulated. Third, it can sustainably express the target protein. Finally, after expressing the target protein, it can be secreted outside the cell. Therefore, our team finally decided to use Pichia to synthesize natural polypeptides, which is also to inherit and further improve the ideas of iGEM teams over the years to explore more possibilities.

Our team then established the engineering bacteria and gene design, and started the initial wet experiment verification, and calculated the yield through the development bands and data of the Western blotting method. The following is a diagram of our technical route, see the steps of the wet experiment (hyperlink to the wet experiment).

After a period of wet experiments, our team found that the expression of our hypoglycemic peptide P37 was unstable in the eukaryotic expression system due to a small molecular weight of only 6.3 kd. So we connected the three VG, with reference to The dipeptide Pro-Asp promotes IGF-1 secretion and expression in hepatocytes by enhancing JAK2 / STAT5 signaling pathway. The Asp-Pro acid-sensitive site mentioned in replaced DDDK enterokinase, because the acid-sensitive site DDDK enterokinase is better controlled and only breaks at PH = 4 to 5, so our team upgraded our gene and added the technical route to adjust PH for acid lysis. Our gene was changed to pPIC9K-3His-Asp-Pro-VG-Asp-Pro-VG-Asp-Pro-VG. The technical route is shown below the following diagram.

On July 14,2024, our team participated in the 11th China iGEMer Exchange Conference (cc). At the meeting, Mr.Xie Ziwen put forward a valuable suggestion to our team: our team needs independent iteration process of innovation. Therefore, our team thought of the fire drug GLP-1 drugs, which are popular for its treatment and prevention of diabetes dependent diseases, especially in the treatment of stroke and cardiovascular disease. Can our drugs also have the function of treating or preventing dependent diseases?

As our team found in our previous background adjustment, the vast majority of patients with diabetes are accompanied by relatively severe disease. CVD risk was 2-to 4-fold higher in diabetic than non-diabetic patients. This is because diabetes is often associated with problems with hypertension, abnormal cholesterol levels, obesity and physical inactivity, which are risk factors for cardiovascular disease. And diabetes mellitus is an important risk factor for stroke. In diabetic patients, atherosclerosis advances more rapidly and often leads to early-onset cardiovascular disease events, which include stroke. Stroke and cardiovascular disease and diabetes is a common dependency, and we conclude by reading the literature, natto kinase has the treatment of stroke and cardiovascular disease, if our hypoglycemic peptide P37 can be hypoglycemic at the same time, the treatment and prevention of dependency from disease, in the current clinical market is more competitive. Moreover, nattokinase and the hypoglycemic peptide P37 have the advantages of medicine / food homology. Compared with the mainstream hypoglycemic drugs on the market, without the huge advantages of any side effects, the efficacy and clinical effect are gradually approaching. Our gene was eventually changed to pPIC9K-3His-Asp-Pro-NK-Asp-Pro-VG-Asp-Pro-VG-Asp-Pro-VG.

Based on the research results of our team, we can through synthetic bioactive peptide P37 into a new type of natural oral peptide drugs for the treatment of diabetes and its complications, through a series of experiments in this laboratory, bioactive peptide P37 has achieved some good research results in the treatment of type 2 diabetes, so we believe that P37 has a potential medicinal value for the treatment of diabetes and complications, P37 food, drug development will breakthrough to solve the treatment and prevention of type 2 diabetes.

Therefore, our team selected backbone Zhangjifan, Zhao Jiayi, Xiao Xinyu, Huang Siyuan and Huang Qiwen to form an entrepreneurial group. Zhang Jia Fan and Zhao Jiayi are responsible for biological experiment and technical support, Xiao Xinyu is responsible for personnel management and staff training, Huang Siyuan is in charge of marketing publicity and software model building, and Huang Qiwen is responsible for market research and publicity in the team. Constitute a team of a company located in its infancy.

When building a corporate culture, guided by the spirit of iGEM, we have made real good and inexpensive hypoglycemic drugs for more than 140 million diabetes patients in our country. At present, the company sets Agrashsolid oral liquid at 188 yuan / box (about 26 USD), and a box can be eaten for a month, with an average of about 6 yuan per day (about 0.83 USD). Combined with the average monthly income of 6072.5 yuan in China in 2023, we fully believe that the vast majority of diabetic patients can afford it. See our business plan (hyperlink) for more details.

As an undergraduate team, our innovation and entrepreneurship ability is insufficient, and the relevant knowledge is still learning. Our team needs training. Our company needs help in technology, personnel and experimental sites and funding. Therefore, our instructor Professor Yao Chenguang introduced to us Liu grong, the boss of Wuhan Lanzhi Biotechnology Co., Ltd. Our team immediately went to Wuhan Lanzhi Biotechnology Co., Ltd.

Liu grong, boss of Wuhan Lanzhi Biotechnology Co., Ltd., warmly received our team. Cheng Si, the leader of our team, first introduced the technical route and product advantages of our project, and emphasized our advantages of using synthetic biology to determine the unique certainty of drug composition, the homology of medicine and food, and reducing production and increasing capacity. After listening to this, boss Liu grong said that our team is committed to independent innovation and the development of domestic new drugs, which was greatly appreciated, and believed that the results of our team are possible for industrial mass production, and agreed to provide experiments and funds for our team.

After completing the project design and making some experimental progress, in order to further determine the feasibility of the project and obtain expert suggestions and feedback, we interviewed Hu Kanghong, director and second-level professor of Sino-German Biomedical Center of Hubei University of Technology. We prepared some questions and got the answers from Professor Hu. These answers and suggestions have greatly promoted the improvement and improvement of our project. His feedback was very positive.

Q1: The hypoglycemic peptide P37 was found in natural soybean, but we used Pichia yeast in genetic engineering to produce P37. What are the advantages of using Pichia yeast compared with natural plant extraction?

A1: The extraction of hypoglycemic peptide P37 from natural plants has an obvious disadvantage, that is, it cannot maintain the uniformity of substance extraction. Soybean in different regions may have different gene sequences, and the quality of drugs cannot be consistent in the process of drug production, which does not meet the standards of drug production. The use of genetic engineering, let Pichia yeast production can maintain the stability of expressed proteins, but also has the advantages of efficient expression, relatively simple operation, suitable for large-scale industrial production. You chose the to to is correct.

Q2: What is your opinion on the competitiveness of P37 in the market after the application of synthetic biology to the hypoglycemic peptide p37?

A2: hypoglycemic peptide P37 is homologous, can be taken orally and non-toxic, plant extraction yield is low and consumption of food, through Pichia production of P37 can greatly improve the production efficiency, save food, in line with the concept of green environmental protection, so the application of synthetic biology to hypoglycemic peptide P37 will greatly enhance the competitiveness of products.

Q3: In the process of constructing the plasmid, we found that the expression of the plasmid of a single vglycin gene was not obvious. Later, we decided to use the series expression of three vglycin genes to increase the expression level. Do you think this method is reasonable?

A3: I think your ideas are reasonable and practical significance. In the production of antibiotics, vaccine research and development, and protein engineering, the technology of serial reexpression of multiple genes may be used. I support your idea, but the biological activity of the produced proteins, etc. I hope you can make further progress.

Q4: What do you think is the most potential future research direction in the field of synthetic biology? What skills or knowledge can we learn at this stage that makes us develop better?

A4: synthetic biology in green energy and environment, medical applications, biological materials and innovation, bioinformatics and other fields show great potential, and your yeast production hypoglycemic peptide P37 project can be classified in biological manufacturing, synthetic biology as a new engine of biological economy, is driving the next generation of biological manufacturing change, through synthetic biology technology build microbial cell factory, can achieve cheap raw materials to high value chemical efficient production, clean and production process of environmental protection. These areas all have great potential. I hope you can find the areas that you are interested in and continue to explore, and ultimately contribute to humanity.

Q5: Our iGEM team is committed to the research and promotion of synthetic biology. What are your suggestions or expectations for our iGEM team?

A5:1, expect you to put forward innovative ideas and solutions in the field of synthetic biology, not only in technology, also included in the application scenarios and business model, team members play their professional advantages, interdisciplinary cooperation, biology, computer science, economics, art can shine in the igm team, looking forward to your project can make a real product.2. You can actively participate in academic exchanges, discipline competitions, and get inspiration in the collision of ideas.3. I hope you can become the disseminator of synthetic biology knowledge, and let more people know about and participate in the research of synthetic biology. I also hope that after the iGEM competition, you can continue to work hard in the field of synthetic biology, turn the team's research results into longer-term research projects or entrepreneurial opportunities, and make contributions to the human society. Finally, I wish you all the excellent results in the iGEM competition and contribute to the development of synthetic biology!

Section 1: Education

1.2 Middle school education

In order to popularize the students' understanding of biology and synthetic biology, our team came to Wuhan Yucai High School and introduced the concept of synthetic biology and the role of synthetic biology in the pharmaceutical industry and maintaining human health to the students.

1.2.1 Introduction to Synthetic biology

Our team started with the knowledge points of fermentation culture in high school biology textbooks, PCR and explained the use of this knowledge in our iGEM project. It can not only help middle school students better understand what they have learned in high school, but also give future students who choose synthetic biology a general direction.

1.2.2 Middle school education feedback

In this activity, in order to make it easier for middle school students to understand, we popularized and figuratively explained all the professional words in the project, explained the basic biology knowledge that middle school students have mastered, and tried to make the explanation more humorous and arouse the middle school students' interest in synthetic biology. Although we get the support of middle school students and high school teachers, but we still found our propaganda method problems, our team project mainly for diabetes drug development, in our propaganda with too much space to promote the iGEM schedule and awards requirements, not considering the middle school students during the university of no concept, our team in this part of the interpretation of the effect is not good, but special interested in our experiment part, especially looking forward to the university biological experiment, we failed to prepare enough video material. Our team will pay more attention to the interest of the target. Despite this flaw, our team still won the first prize of Hubei University of Technology in Wuhan Yucai Senior High School.