In order to better fit the theme of human practice, our team hopes to view our project from multiple aspects and guide our project design. In order to understand the current social situation, we learned about experts' suggestions and patients' needs through various surveys including questionnaires and interviews. At the same time, you can learn about existing relevant product information by checking online information. We have also envisioned effective measures regarding possible feedback from society. We hope that our connection with society will be a virtuous cycle of mutual benefit and support the continued improvement and development of relevant research. Finally, through summary, we present our experiences and thoughts in this section.

Recently, we saw a video about the impact of cholesterol in food on the human body. The example in this video mentioned eggs, a food that people eat every day.

source:https://mp.weixin.qq.com/s/Xc-b2ffg90DSxnjo0fDp8g
This makes us want to pay attention to the total cholesterol intake in our daily diet and the harm of cholesterol to the human body. So we tried to look up some information. We learned that eggs are rich in lecithin, protein, calcium, iron, phosphorus and other substances. This can not only replenish the energy consumed by the human body, but also serve as the body's nutritional reserve, participate in the body's metabolism, repair human tissues, and improve the body's immunity. However, eggs do contain a certain amount of cholesterol, and eating too many eggs will certainly lead to high cholesterol.

The literature states that each 50g egg contains 186mg of cholesterol. This is much higher than the cholesterol content in other foods. (The Impact of Egg Nutrient Composition and Its Consumption on Cholesterol Homeostasis Heqian Kuang, Fang Yang, Yan Zhang, Tiannan Wang, 1 and Guoxun Chen Published online 2018 Aug 23. doi: 10.1155/2018/6303810). Likewise, too much cholesterol can lead to an increased risk of some diseases. Literature in Circulation shows that eating one egg a day significantly increases the risk of death from heart disease. Higher blood cholesterol levels and higher dietary cholesterol intake are also associated with an increased risk of death from heart disease. (Zhao B, Gan L, Graubard BI, Männistö S, Albanes D, Huang J. Associations of dietary cholesterol, serum cholesterol, and egg consumption with overall and cause-specific mortality, and systematic review and updated meta-analysis. Circulation. Published online April 1, 2022. doi:10.1161/CIRCULATIONAHA.121.057642) At the same time, high cholesterol can also cause other diseases such as high blood pressure, diabetes, etc.
We want to solve the problem of high cholesterol, and we invited a biomedical doctor to have an online discussion to discuss our project

To sum up, our goal is to design a probiotic product that the human body can absorb and degrade cholesterol to help those with high blood cholesterol levels reduce cholesterol intake and prevent the occurrence of cholesterol-related diseases.

1. Project background investigation

The initial idea of our team was to use in vivo microbial therapy to allow microorganisms to react in the human intestine. Then we learned about China’s biosafety laws.

Source: https://www.mee.gov.cn/ywgz/fgbz/fl/202303/t20230314_1019536.shtml
We found that allowing E. coli to react in the human intestine poses a huge biosafety problem. At the same time, China’s national regulations are strict and it is not easy to approve the production of engineered probiotics that work in the human intestine. Therefore, we decided to use bacterial powder used outside the human body. , use bacterial powder to process food in factories and directly produce low-cholesterol food.

2. ACS gene and ismA gene

At the beginning, we used the acs gene. The literature shows that the acs gene can degrade cholesterol. The ACS gene can combine different fatty acids with CoA to form the corresponding fatty acid-CoA, thus promoting the anaerobic cholesterol degradation process. However, the experimental results were not ideal, so we reviewed the literature and had an online discussion with Teacher Liu.

Teacher Liu said that because acs cannot participate in the first step of cholesterol conversion, this gene will not have a very good effect in vitro, so we looked for a gene that can directly degrade cholesterol. We found the isma gene, and the literature shows that it can directly degrade cholesterol. cholesterol.
We know that after the human body ingests cholesterol, intestinal microorganisms can directly convert it into Coprostanol, which cannot be absorbed by the human body, through a series of metabolic reactions. In this transformation process, the IsmA gene plays an important role. It encodes a hydroxysteroid dehydrogenase (HSD) and is involved in the first and last steps of the transformation process. The IsmA gene can well promote the conversion of cholesterol, thereby reducing cholesterol levels in food.
We decided to use the ismA gene.
 

3． Oleic acid promoter

Our team members had online contact with nutrition expert Teacher Gao. He told us that oleic acid has the effect of degrading cholesterol and is good for the human body. Inspired by this, we hope to combine our topic with oleic acid, so we designed an oleic acid promoter. We hope that adding oleic acid can activate the expression of subsequent genes, and that consuming oleic acid can also degrade cholesterol. Moreover, during the fermentation stage of the factory, the strain will not produce oleic acid because there is no oleic acid in the factory, and subsequent genes will not be expressed, which can reduce the pressure on the bacteria. Therefore we chose to use the oleic acid promoter

4. GalU gene

The acs gene cannot participate in the first step of cholesterol conversion, so this gene will not have a very good effect in vitro. We asked Dr. Yin of Shanghai Jiao Tong University, and Dr. Yin suggested the GalU gene for us.

5. EPS

We reviewed the literature and found that EPS can adsorb cholesterol. At the same time, we had discussions with two other IGEM teams, and we found that they were also trying to use EPS, so our three teams held an online discussion.

Source: https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1348-0421.2010.00219.x
We found that the strain's EPS production capacity and cholesterol removal rate were significantly correlated. Generally speaking, strains that produce large amounts of EPS can remove more cholesterol from the culture medium than strains that produce low EPS, and the GalU gene can enhance EPS production, so we decided to use the GalU gene.

6． T7 promoter

We had an online discussion with the teacher. After discussion, we decided to put the transporter under t7 and the others under the transporter, so that cholesterol can be transported into the bacteria immediately, with faster transport speed and higher efficiency.

7. Overall genetic circuit

Our overall genetic circuit is as follows:

At the beginning of the project, we wanted the powder to enter the body for action. In addition to reviewing the information, we also asked Lawyer Cai for his opinion.

We have two methods to achieve in vitro effects. The first method is to produce powder and add it to the food for patients to eat; the other is to send the powder to food processing plants for production and processing to produce semi-finished products or The finished food is given to patients.
Because this product is intended for use by cholesterol patients, we conducted interviews with cholesterol patients to understand their preferences and opinions. Most patients prefer the second method, which they believe is more convenient and safer.

Of course, because cholesterol can cause many cardiovascular diseases, and the rate of cholesterol is also related to the incidence of underlying diseases such as diabetes, many healthy people are also interested in our products to prevent cholesterol in advance [https://onlinelibrary. wiley.com/doi/10.1111/jdi.13316]
We also conducted street interviews to understand people’s opinions on our products

We conducted a questionnaire survey on this issue. A total of 82 people participated in the survey, 39% were cholesterol patients, 41% were healthy people, and 20% were unclear. 72% are willing to try our products, 15% are unwilling, and 13% need to know more.
But from the overall data, most healthy people and cholesterol patients are willing to try our products.

As the low-cholesterol foods co-produced with the help of the foundry gradually enter the market and are accepted by the majority of users, we undoubtedly expect this product to contribute to public health in the future. First, we hope that this food can play an important role in cardiovascular health and become one of the effective options for helping people lower their cholesterol levels. As more people choose low-cholesterol foods, we expect to see more health benefits and a positive response in the coming years. We will also later iterate on the food to improve its low cholesterol-lowering effect. At the same time, the application prospects of bacteria powder that directly acts on the body will still be one of our goals for continued research and improvement in the future. We hope to be able to actually apply our research in this area in the future so that people with high cholesterol will have more diverse choices.
We also believe that technology applied to food will most likely face social criticism, and some people will have reservations about this new type of food. Therefore, we will work closely with regulatory agencies and health experts in advance, use rigorous scientific research and clinical trials to prove its safety and effectiveness, and continue to provide scientific basis to the public.
Overall, our outlook and expectations for the future of low-cholesterol foods are positive. Through continued efforts and innovation, we believe this food will make great improvements to public health and bring health and well-being to more people. At the same time, we will actively listen to social feedback, continue to improve and enhance our products, and continue to make our own efforts for public health and people's happiness.