In the project of the Guangzhou-GEC team, we focus on the targeted treatment of colorectal cancer through engineered probiotics. Human Practice plays an important role in the development of the project, especially Integrated Human Practice, which supports our work in and outside the laboratory and has a positive impact on project design and laboratory operations. In this process, we focused on two main elements, namely project promotion and feedback from relevant stakeholder groups. We adhered to humanistic ideas to promote the project and conducted in-depth interviews with herbalists, stakeholders, potential users, and professional herbalists/experts.Through interactions with these key individuals, we were able to further improve and revise our project. We have invested a lot of time, energy and resources in contacting experts, patients, and pharmaceutical companies online, attending exchange meetings, joining patient associations, researching literature, and even registering in hospitals just to ask doctors about the true situation of colorectal cancer patients. These experiences provided opportunities to deepen our knowledge of the condition while receiving structured feedback on the project from expert patients. Taken together, this process provides us with key information to help us better shape the project, ensuring that we do not deviate from the right direction during the project development process, so that we can better apply our project to the real world.

When exploring the direction of the project, we were inspired by the story of one of the team members, Xiao Zhang: his janitor was a kind and enthusiastic person who was especially kind to Xiao Zhang. But one day, he suddenly disappeared. After asking his neighbors, he learned that he had colorectal cancer due to irregular eating and excessive drinking. Because the early symptoms of colorectal cancer were not obvious, and he neglected his health problems and never had regular physical examinations, the disease had entered the mid-to-late stage. He had always taken good care of Xiao Zhang, and Xiao Zhang was thinking about him, so he and his mother decided to visit him. When he arrived at the hospital, he discovered that he was undergoing chemotherapy, and the side effects of the chemotherapy drugs made him in a very bad condition.After talking to the doctor, I learned that the current conventional treatments include surgery, chemotherapy, etc., and the doctor used a chemotherapy drug called 5-fluorouracil for him. We then further understood the therapeutic principle of this drug, and roughly understood that it affects cell growth by disrupting the nucleic acid metabolism of tumor cells. However, in addition to affecting tumor cells, it will also affect the normal replication and division of other cells in the human body, resulting in a very negative effect of this chemotherapy drug. Through understanding, we imagine that if this chemotherapy drug (pentafluorouracil) can be targeted to treat colorectal cancer, the occurrence of side effects will be greatly reduced. Therefore, we gathered a group of classmates who also loved biology and formed a team. We hope to use the power of synthetic biology to complete this idea.

Format: Offline group meetingKey point: After several rounds of group discussions, we realized that we still don’t know enough about colorectal cancer and its patients, so after discussion, we planned to divide into two groups: the first group is responsible for receiving information from oncology and anorectal doctors. Acquire professional knowledge about colorectal cancer; the second group is responsible for communicating with the patients' families about the most direct situation of the patients.

Format: Offline hospital registration and consultationKey point: In order to have a further professional understanding of colorectal cancer, the first team members came to the hospital to consult with the anorectal surgeon. The doctor briefly introduced the symptoms and stages of colorectal cancer to us, as well as some expanded knowledge about colorectal cancer.Specific: In the first group, based on relevant reports provided by several doctors, we have a better understanding of colorectal cancer - colorectal cancer is a common malignant tumor in the gastrointestinal tract, and its early onset is mainly related to European countries and is a disease of affluence. A sort of. With the rapid development of China's economy, employment opportunities have generally increased, international trade has increased, and population movements have become more frequent, which has led to a substantial increase in the incidence and mortality of colorectal cancer in China. The early symptoms of colorectal cancer are not obvious. As the cancer grows, symptoms such as changes in bowel habits, bloody stools, diarrhea, alternating diarrhea and constipation, and local abdominal pain appear. In the late stages, systemic symptoms such as anemia and weight loss appear. Its morbidity and mortality rate are second only to gastric cancer, esophageal cancer and primary liver cancer among digestive system malignant tumors.Conclusion: We can learn more about colorectal cancer more effectively by asking doctors for advice.

Format: Online group chatKey point: In order to understand the most direct situation of colorectal cancer patients and their families, members of the second group joined the patient association group chat of colorectal cancer patients and their families on the "Little Red Book" platform. Through the chat records in the group, we learned that Patients with colorectal cancer will choose surgery, chemotherapy and traditional Chinese medicine for treatment. And most people's views are "if you can do it without chemotherapy, don't do it. Chemotherapy is very harmful to the body", "Chinese medicine is very good for conditioning" and so on. By joining the patient association this time, we have a better understanding of the voices of patients and their families. We also understand that most people are treated with surgery and chemotherapy, and some people also receive traditional Chinese medicine. Among them, traditional Chinese medicine conditioning is a treatment method that we have never thought of before. This discovery allows us to think more about the treatment of colorectal cancer.Conclusion: Joining the patients will give us the most intuitive understanding of the treatment methods for colorectal cancer patients and their families, feel the voices of patients and their families, and allow us to think about more aspects of treatment methods.

Patient group chat history

Format: Online viewingKey point: By reviewing the literature, we found that 5-fluorouracil (5-FU), a typical chemotherapy drug, can be produced from a non-toxic prodrug, 5-fluorocytosine (5-FC), via cytosine deaminase (CD).Conclusion: It is understood that pentafluorocytosine (5-FC) can be converted into pentfluorouracil by cytosine deaminase (CD). We further thought: with this conversion, 5-fluorouracil will not directly play a role in the human body, thereby reducing the impact of 5-fluorouracil on normal cells except cancer cells. This discovery greatly encouraged us.

Format: Online consultationKey Point: We shared our findings from the literature survey with our teachers and asked for help on how to make this conversion happen in humans. The teacher proposed that microbial therapy can be used to achieve the transformation of pentofluorouracil in the human body.Conclusion: With the help of biology teachers, we applied microbial therapy to make drug conversion occur in the human body.

Chat history

Format: Online meetingKey points: We introduced the project concept to Dr. Sun in detail. First of all, Dr. Sun said that he deeply felt that it was not easy for the team to produce results. He also encouraged us to have the courage to challenge research when we were still high school students and hoped that we would continue to persevere. Secondly, Dr. Sun is highly positive about the overall concept and ideas of our project and believes that compared with traditional injection treatments, our project is more innovative.Conclusion: Our project has been affirmed by oncologist Dr. Sun. Dr. Sun believes that compared with traditional injection treatment, the use of suppositories is more innovative.

Format: online communicationKey points: We introduced the project concept to colorectal cancer patients in detail, showed Dr. Sun’s affirmation of our project, and asked the patients whether they would accept this novel treatment method using genetic engineering technology to modify E. coli. After the patient roughly understood our treatment method, he said to us, "This treatment method does seem to reduce a lot of side effects, but we don't know anything about the professional knowledge. You are high school students, but I see you have a hospital director." To ensure the safety of the treatment, we are still willing to try it.”Conclusion: Colorectal cancer patients expressed that they would try to use our treatment method (genetic engineering technology to modify chassis microorganisms) after ensuring the safety of the project.

Format: Offline exchange meetingKey point: During the CCIC exchange meeting, we communicated with the team from China Medical University and found that they planned to use E. coli Nissle1917 to treat colorectal cancer. After they explained to us the reasons and benefits of choosing this strain, we decided to also use Nissle1917 as the chassis microorganism for our project.Specifically: Escherichia coli Nissle1917, referred to as ECN, is the only species of Escherichia coli that is not pathogenic and is a probiotic.The mechanism of action of ECN is that it can colonize the human intestine and prevent pathogenic bacteria from invading the intestinal mucosa, and has a protective and repairing effect on the intestinal mucosal barrier. EcN also participates in the immune regulation of the host body, balances the secretion of immune factors, enhances the host's immune ability, and thereby alleviates and treats inflammation. The latest research has found that ECN has a tumor-targeting effect, and its combination with chemotherapy drugs can enhance the anti-tumor efficacy of the drugs.Conclusion: Through offline exchange meetings and sharing with other teams, we determined the application of chassis microorganism Nissle1917.

Format: offlineKey point: After interviewing Dr. Li from Sun Yat-sen University in the field of biology, we gained new inspiration. Dr. Li pointed out that if we can focus the conversion of 5-fluorouracil around cancer cells, the efficacy may be improved. This idea was inspiring to us, and we realized that targeting cancer cells might be the key to improving efficacy.Conclusion: Through Dr. Li’s advice, we realized that the targeting system is the key to improving the efficacy.

Format: Online viewingKey takeaway: After poring over a lot of literature, we found a specific protein that we thought could help us achieve what we were targeting. Therefore, we decided to introduce this protein into our second-generation product system in order to achieve more precise targeted therapy.Details: During the carcinogenesis of colorectal cancer cells, HSPG protein is highly expressed, so the HIpA protein targets the engineered strain around the cancer cells by binding to HSPG (heparin sulfate proteoglycan) on the surface of tumor cells. Targeting engineered strains to treat colorectal cancer.Conclusion: Select the HIpA protein gene to achieve targeted therapy.

Source: https://doi.org/10.1038/s41551-017-0181-y

Format: onlineKey points: We contacted a specialized pharmaceutical company through the Internet, introduced the project concept and current improvements to your company in detail, and hoped that your company would give you some suggestions. Your company pointed out the shortcomings of our project - enzymes in the engineered bacteria will affect the conversion of drugs. And proposed the use of cell surface technology to solve this shortcoming, detailing what cell surface display technology is. We are very grateful to accept your company's improvement guidance.Specifically: Cell surface display technology is a biotechnology that expresses and localizes target proteins on the surface of microbial cells, also known as microbial surface display technology. This technology enables researchers to manipulate and change proteins directly on the surface of biological cells, making it easier to study biochemical and biophysical properties, enzyme catalytic reactions, and cell engineering.Conclusion: The pharmaceutical company proposed the use of cell surface display technology. We can display our enzymes on the surface of engineered bacteria, which allowed us to further improve the project.

In the targeted therapy module, cell surface display technology: ice nucleation protein (INP) is used to anchor the HIpA protein on the surface of the strain. During the carcinogenesis of colorectal cancer cells, HSPG protein is highly expressed, so HIpA protein targets and anchors to the surface of cancer cells by binding to HSPG (heparin sulfate proteoglycan) on the surface of tumor cells.Name introductionThe core of this technology is the use of naturally occurring signal peptides or carrier proteins that can localize proteins to the cell surface. Usually these proteins transport proteins to the cell surface through specific pathways. In engineering biology, we can use genetic engineering methods to connect the gene sequence of the target protein to the gene sequence of these signal peptides or carrier proteins to form a fusion gene, and then introduce the fusion gene into microorganisms so that they can be expressed and expressed in cells. localized to the cell surface.Ice Nucleation Protein (INP, Ice Nucleation Protein)Ice Nucleation Protein (INP) is a protein carrier commonly used in cell surface display technology. The INP protein has a large domain outside the cell that can be used for surface display of other proteins.

Format: Online group meetingKey point: In this group meeting, we discussed and designed the dosage form of the product. During the initial design, we originally planned to make the final product into bacterial powder for administration, but this idea was quickly rejected by many members. The main reason is that the bacterial powder is easily corroded by gastric acid. The live bacteria are not evenly distributed and are difficult to preserve once the package is opened and oxygen enters. So we improved the product into capsule delivery.Conclusion: The group meeting discussed changing the dosage form of the product from bacterial powder to capsules.

Format: information review/group discussion

(Source: https://mp.weixin.qq.com/s/NirZ3c9idmmO2G6EexHoDw)Key point: Through data survey, we have a more realistic understanding of the pain points of colorectal cancer patients and their families - patients with mid-to-late stage colorectal cancer have inconveniences in daily life, eating difficulties and other inconveniences due to the disease. Making it difficult to take orally. After group comparison and screening, we finally transformed the product into a suppository form.Specific: Oral vs SuppositorySuppository: Only one dose is needed, it can reach the lesion directly, and it is highly practical.Oral administration: Two doses are required. Oral administration will consider issues such as gastric acid corrosion and inconvenience for patients in the middle and late stages.Conclusion: After group discussion, the dosage form of the product was finally finalized as suppositories.

Format: Offline exchange meetingKey point: In July this year, our team was invited by the CICC Organizing Committee of the China IGEM Division to participate in the largest academic seminar and exchange meeting in the China IGEM Division held in Hainan. During the meeting, we listened carefully to the introductions of their projects by other university student teams. Our team benefited a lot from the diversified project development directions and many unconstrained ideas, and these teams all had a common goal. Characteristics, attention and vigilance to biosecurity. Many of their approaches to the experimental process were eye-opening for us. During the continuous sharing process in the meeting, our team continued to improve its design plan and solved many safety hazards in previous designs. After the meeting, our team started private group project exchanges with two teams from the Pharmaceutical University and Beijing with new ideas. These two university student teams have similar research directions to ours. Their rich university experience and experimental experience have provided our team with many valuable safety case sharings, which not only broadened the depth of our biological knowledge, but also comprehensively expanded our Our breadth of knowledge paved the way for successful and safe subsequent experiments.Conclusion: At the CCIC exchange meeting, I learned to communicate with other teams, improved my knowledge, and gained a better understanding of project safety.

Before using the medicine, you need to wash the perineum, anus and hands with clean water. If the package contains a finger cot, you can put the disposable finger cot on the index finger, remove the outer packaging, take out the suppository, put the cone head toward the anus, gently insert it, and use Use the index finger covered with a finger cot to push the suppository 6-7cm into the rectum.To prevent the suppository from slipping out of the anus, you need to lie on your side when inserting the medicine, and stay in bed for at least 15 minutes after inserting to facilitate the diffusion and absorption of the medicine.

Format: Online discussion/online communicationKey point: When the project was basically taking shape, the team members conducted reflection and review. We analyzed the causes of colorectal cancer based on the cases of Uncle Zhang and other patients with colorectal cancer, and found that poor eating habits are one of the important causes of colorectal cancer. Foods high in fat, high in protein, low in fiber, high in calories, excessive alcohol consumption, and containing carcinogens such as nitrosamines will increase the risk of colorectal cancer. In order to prevent and better treat colorectal cancer, we have specially designed a healthy and nutritious recipe in accordance with the National Healthy Diet Law. And it is published and promoted online to protect the health of more people.