As a new team in iGEM of the School of Basic Medicine, we spent several weeks learning about the iGEM competition in detail under the guidance of the advisor who had won the gold medal. We learned that this competition aims to use synthetic biology to solve human problems.

Although synthetic biology can be applied to many topics, based on the expertise of team members and PIs, we ultimately decided to choose between the diagnosis and treatment tracks. Since many first-year students are on our team and the foundation required for synthetic biology is still relatively weak, PI Professor Zhang Ling led everyone to carry out the winter camp.

Students from the School of Basic Medicine, Bethune Third Clinical School of Medicine, Bethune School of Stomatology, 19 students from the Class of 2020, Class of 2021, and Class of 2022 from five colleges, including the School of Life Sciences and the School of Mechanical and Aerospace Engineering are engaged in online training around the clock. We will then brainstorm to pursue our project goals and find out which ideas are reasonable and have the potential to go global and benefit the world.

In the winter camp, we learned relevant knowledge about synthetic biology, including using the iGEM official website, basics of synthetic biology, literature search, medical professional English, molecular biology, cell biology, and microbiology. We learned about gene editing technologies, such as the CRISPR-Cas9 system, and how these technologies can be used to build more complex biological systems. We also learned how to use bioinformatics tools to analyze and understand data from biological systems. This gave us a deeper understanding of the theory and application of synthetic biology and laid a solid foundation for future scientific research.

In subsequent brainstorming sessions, we came up with countless inspirations, from fighting multiple sclerosis to early warning of tumor cells. The PI suggested that we focus on our perspectives and conduct more in-depth literature research. At the same time, based on PI's current academic research mainly focusing on cancer, we can get more professional guidance and advice. Finally, we focus on cancer and look forward to applying synthetic biology to solve some of the current bottlenecks of cancer.

Based on the conversation with the PI, we decided to conduct further research on cancer as a next step. We will conduct in-depth literature searches and analysis of previous teams to find a highly innovative and feasible solution. After that, we will present our findings in the next group meeting to find a suitable synthetic biology method.

Our team has gone through many brainstorming sessions, and everyone has conducted in-depth literature searches and analyzed previous outstanding sections. We look forward to professors providing us with feedback and guidance to determine a more precise route for our project.

In the second meeting with the PIs, many students mentioned genetic engineering to ensure biological safety. Among them, the application of suicide genes stands out.
"Suicide gene" therapy is to transfect a gene encoding a specific enzyme (suicide gene) into tumor cells and then use drugs to kill the cells. This drug is non-toxic to normal cells but has a selective killing effect on tumor cells because the enzyme encoded by the introduced "suicide gene" converts the drug into substances harmful to tumors, making the tumor cells unable to replicate their DNA and die.
At the same time, nucleic acid interference drugs have gradually entered people's field of vision in recent years. PI introduced RNA interference (RNAi) technology to us in detail: RNAi is a process that can specifically and selectively destroy the expression of target genes. Among them, dsRNA molecules can efficiently and specifically inhibit mRNA transcription, promote mRNA degradation, and thereby inhibit the expression of specific genes.
Small nucleic acid drugs based on RNA interference technology can silence the expression of oncogenes, have good drug potential, and can treat both symptoms and root causes. The three mechanisms of small nucleic acid drugs, which specifically kill cancer cells and stimulate the body's immune response, exert anti-cancer effects. They have the characteristics of reasonable safety, high targeting, broad anti-cancer spectrum, and strong killing power against cancer cells. They can effectively treat a variety of cancer cells. Solid tumors.
Through conversations with the PIs, we initially determined that the team would apply RNAi technology for cancer treatment.

Based on the PIs’ suggestions and discussions among the team, we decided to continue to study RNAi interference technology in depth and study the literature to determine the vector. After finding a suitable carrier, our technical theme will be roughly determined, thus reaching the first milestone!

Today, we have roughly determined that we will use RNA interference technology to solve the cancer problem, and the choice of vector is the problem we face today. Typical vectors include plasmid vectors and viral vectors.

How to choose? We again made an appointment with the PIs and the instructor, who is familiar with RNAi technology, hoping they can provide some professional opinions.

Cong Cong, who is familiar with experimental techniques, pointed out that plasmids usually have excellent stability and are not sensitive to temperature. Viruses are different. They generally need to be stored at low temperatures and are very sensitive to temperature. Repeated freezing and thawing also have a significant impact on viability. Moreover, plasmid vectors have a large carrying capacity, are easy to operate, and are relatively cheap. We aim to apply new technologies to benefit more people, so universality and economy are essential. Therefore, plasmids are undoubtedly our best choice. The PI also explained that chemically synthesized siRNA has a short half-life in the body, is easily degraded, and needs to be administered continuously during treatment.

The shRNA expression system can self-replicate in tumor cells and continually express shRNA-silencing genes. The PIs suggested that we can choose to construct shRNA plasmids to conduct our research. Cong Cong, who is familiar with experimental techniques, pointed out that plasmids usually have excellent stability and are not sensitive to temperature. Viruses are different. They generally need to be stored at low temperatures and are very sensitive to temperature. Repeated freezing and thawing also have a significant impact on viability.

Moreover, plasmid vectors have a large carrying capacity, are easy to operate, and are relatively cheap. We aim to apply new technologies to benefit more people, so universality and economy are fundamental. Therefore, plasmids are undoubtedly our best choice. The PI also explained that chemically synthesized siRNA has a short half-life in the body, is easily degraded, and needs to be administered continuously during treatment.

The shRNA expression system can self-replicate in tumor cells and constantly express shRNA-silencing genes. The instructor suggested that we can choose to construct shRNA plasmids to conduct our research.

After professional guidance, we finally constructed a shRNA plasmid to silence oncogene expression. Initially, we chose the Therapeutics, looking forward to using this synthetic biology approach to treat cancer. Next, we will conduct in-depth research on how to deliver plasmids into the body to achieve a highly innovative and rational method and determine the use of plasmids as vectors rather than viruses. The project track has been initially chosen as the Therapeutics!

After previous discussions, our team conducted a series of meetings. It was thought that liposomes might be used for drug delivery. Since PI. Yang is an expert in molecular biology, we approached him this time and hoped he could make professional suggestions on the plasmid delivery system. Propose a feasible solution for the implementation of our RNAi technology.

At the meeting, PI. Yang immediately rejected our idea about liposomes. He proposed that the toxicity of liposomes is too significant for pharmaceuticals and that clinical administration requires continuous administration.

When liposomes are used as carriers, they have low stability. They are easily degraded by enzymes in the blood, causing the loaded plasmid to be released too quickly and reducing the therapeutic effect. At the same time, liposomes have limited loading capacity and cannot carry large amounts of plasmids, limiting therapeutic drug payload and requiring continuous administration. Moreover, liposomes have limited recognition and uptake efficiency in targeting tumor cells, resulting in low drug concentrations in tumor tissues. It lacks targeting when administered intravenously, which can lead to adverse effects such as hand-foot syndrome.

As for implementing our project, PI. Yang gave his reliable suggestions. He proposed that we can focus on the transformation of engineering bacteria and achieve cancer treatment through the change of engineering bacteria. Based on PI. Yang 's research, he suggested that we might be able to use attenuated Salmonella to realize our idea.

We immediately thought that we had learned about bacterial therapy during a previous literature search. Bacteria-mediated cancer therapy, BMCT can be highly targeted, powerfully anti-tumor, improve the tumor immunosuppressive environment, effectively inhibit cancer metastasis and recurrence, serve as a drug delivery system, overcome drug resistance and enhance the effect of combination therapy, etc., bringing new possibilities and hopes to cancer treatment.

He also pointed out that Salmonella has the advantages of high tumor specificity, deep tissue penetration, natural bacterial toxicity, convenient genetic modification, and exemplary safety. Furthermore, Salmonella has intrinsic anti-tumor activity. Salmonella not only has excellent advantages as a carrier; it can deliver plasmids into tumor cells, but it can also trigger immune responses, both of which have anti-tumor activity and enhance the effect of our plasmids in treating tumors.

We decided to introduce the plasmid into Salmonella and use Salmonella as a vector to deliver the plasmid. After discussion, we decided to obtain attenuated Salmonella through genetic engineering technology and use the characteristics of attenuated Salmonella to have no noticeable side effects, overcome the penetration limitations of tumor tissue, trigger immune responses, and induce anti-tumor immunity to carry out our project. Next, we will find a suitable Salmonella strain through an in-depth literature search and professional professors.

PI presented the outstanding advantages of Attenuated Salmonella pq in several strains, indicating that it was promising and possible to research cancer universality.

After determining the use of Salmonella, we began an extensive search for information, hoping to find a strain that had been proven to be both attenuated for modification and had the potential to be innovative. After selecting several systems, we contacted our PI for another meeting to get professional advice.

The PI noted that although numerous scholars had modified Salmonella at present, the basic logic was broadly similar. TCSs, Two-Component Systems, are the most crucial information transfer systems of bacteria, capable of transferring unfavorable environmental conditions external to the bacteria.

The PhoP-PhoQ system of Salmonella is one of the classical two-component systems, which consists of PhoQ, a sensor protein, and PhoP, a reaction regulatory protein. The PhoP-PhoQ two-component system enables Salmonella to sense changes in the external microenvironment, such as H+ antimicrobial skin and divalent cations, and directly or indirectly coordinates the expression of hundreds of virulence genes to resist predation by the host immune system and to survive and multiply in phagocytic vesicles. (Mutants reduce Salmonella virulence to colonize the strain in vivo).

The Pho-PhoQ two-component system regulates the expression of about 7% of the genes in Salmonella pq. Deletion of either PhoP or PhoQ or an imbalance in the trans phosphorylation process between Phop-hoQ due to mutations in the coding region of PhoQ can reduce the virulence of Salmonella pq. The Pho-PhoQ two-component system regulates the expression of around 7% of genes in Salmonella. Deleting either PhoP or PhoQ or mutations in the PhoQ coding region that disrupt the phosphorylation process between PhoP and PhoQ can diminish the virulence of Salmonella. Since our principal investigator (PI) has already researched the attenuated Salmonella strain pq, we have decided to embark on fresh explorations in this field. The impressive performance of the attenuated Salmonella strain pq, particularly in terms of its anti-tumor capabilities, has bolstered our confidence in its potential for cancer therapy.

Through communication with our laboratory teachers, we have learned that the idea of conducting preliminary research experiments for a universal cancer therapy is feasible. Upon researching, we discovered a novel anti-cancer approach involving the inhibition of the PD-1/PD-L1 signaling pathway, which has proven successful in combating nearly 20 central solid tumors, including lung cancer, gastrointestinal tumors, breast cancer, urological tumors, skin cancer, and lymphoma. This approach has substantially improved the survival rates of late-stage cancer patients, making it a "breakthrough" treatment for cancer patients. The medical community and patients have unequivocally witnessed the profound transformation brought about by the era of immunotherapy in late-stage cancer treatment.

Following this conversation, we have clarified the use of Attenuated Salmonella strain pq. Simultaneously, we have strengthened our confidence in researching a universal cancer therapy and have initiated preliminary experiments in the laboratory, ensuring both biosafety and public health considerations. We intend to conduct comprehensive research on various cancer treatments. This marks a new milestone for us!

Once we established the bacterial strain's usage and decided to embark on universal cancer research with it, we decided to seek out an expert with in-depth expertise in cancer treatment. We aimed to discuss our ideas with them and acquire professional guidance.

After listening to our ideas, Prof. Jing Li immediately said we should not aim too high and directly pursue universal cancer research. She emphasized that, given our current level of experimentation and the available time, attempting universal examination or investigating multiple types of cancer simultaneously is not a practical approach. She suggested that we focus on our goals and study a specific type of cancer. Given that the process involving the inhibition of the PD-1/PD-L1 signaling pathway to induce cancer cell death has shown progress in various cancer types, our team, after in-depth discussions and data collection, decided to utilize Salmonella as a carrier for cancer treatment in only one or two specific cancer types. This strategy is aimed at improving research efficiency and feasibility.

Finally, our focus narrowed down to lung cancer and colorectal cancer. Lung cancer, primarily non-small cell lung cancer, is among the most common cancers globally. According to 2018 global statistics, it ranks first in incidence and mortality among men and second among women. Due to its high incidence and mortality rates, we chose it as one of our treatment directions and considered the possibility of aerosolized inhalation delivery. Simultaneously, colorectal cancer, which in 2020 became the fourth most common malignancy, is characterized by limited cure rates with conventional treatment methods and the occurrence of multiple side effects. Since Salmonella is a part of the gut microbiota and might possess better colonization capabilities in the intestines, we have opted to use Salmonella for the treatment of colorectal cancer, considering the possibility of oral administration.

We changed our research idea after listening to Ms. Jing Li's suggestion. After communicating with our lab instructors。, we plan to conduct research on two types of cancer, colorectal cancer and lung cancer, simultaneously. Since the same strain is used, the difficulty of the preliminary work is not too big! Next, we will evaluate various aspects and try to focus our perspective on one type of cancer as soon as possible.

After determining our initial research direction, we began to seek different suggestions from various parties to perfect our experimental plan. We found Dr. Kewei Ma, a lung cancer treatment expert in the Department of Oncology of the First Hospital of Jilin University, in the hospital to share our ideas with him and interview him. We asked him for his advice on microbial therapy, a new type of therapy, and how to administer it.

Dr. Kewei Ma is skeptical of bacterial therapy since the drug needs to stimulate the body's immune system. Still, the immune system is weakened in patients with cancer, and the current process has no assessment of the immune system. And he raised objections to nebulized drug delivery. Although nebulized therapy uses less medication, delivers it accurately, and has a quick onset of action, it has high toxicity and side effects, and patients with weak sputum expectoration may have increased inflammation, and prolonged nebulization may also increase cardiac burden. In the case of improper operation, nebulization may harm the lungs, including inducing bronchospasm, respiratory tract infection, airway burns, and so on.

Moreover, lung cancer is a kind of malignant tumor with a certain degree of consumptive nature, and patients may experience a decline in body resistance during the treatment of the disease. And nebulization treatment at this time may aggravate the above situation, which is not conducive to the recovery of the disease. Nebulized inhalation may also cause a series of complications, such as allergic reactions, aggravation of wheezing symptoms, with varying degrees of fever, irritation of infections, aggravation of the symptoms of chest tightness, shortness of breath and dyspnea, nausea and vomiting, dry mouth and eyes and other uncomfortable symptoms. At the same time, nebulized treatment can generally only give small molecules of drugs. Bacteria are too large and are usually not offered through nebulization.

After learning about the goals of our competition, he recommended that we visit the FDA to seek further information about biosafety.

Through this exchange, the treatment of lung cancer had been questioned. But we don't want to give up yet! We decided to contact an expertise in biosafety to further improve our program to ensure that safety is maximized.

At present, we have communicated with clinicians, researchers, technology companies, etc. Before conducting further experiments, we hope to share with teachers from the school ethics committee to understand whether the team's idea can be realized ethically and what problems we must pay attention to during the experiment.

In the communication with Prof. Liu, we learned that the state has issued corresponding laws, regulations, and review methods for biosafety and ethics. For example, in the laboratory, we should strengthen the management of experimental raw materials, maintain the inspection and maintenance of laboratory equipment, and inform us which practical wastes need harmless treatment. At the same time, each interviewee should be respected in terms of ethics.

At the same time, in the questionnaire or survey process, the respondents' informed consent should be obtained, and their privacy information should not be disclosed. Stakeholders should know the meaning of the questionnaire and what data the team needs to use, which provides a fundamental guarantee for us to distribute the questionnaire.

Following discussions with Prof. Haiyan Liu, we have addressed the issues that demand our attention in our experiments. Under the guidance of Prof. Liu, we actively researched laws and regulations about biosafety and ethics. Concurrently, we initiated the drafting of informed consent documents and privacy protection guidelines to ensure the privacy and security of respondents during the survey process. Our project is now infused with renewed confidence! Our next step is to seek new experts' assistance regarding our experiments' safety and rationality.

By then, our team has already set our sights on using the RNAi mechanism to silence the gene that may assist cancer development. Nonetheless, the selection of the specific gene to degrade remained unclear. Though all information retrieved from previous iGEM teams and relevant articles are thoroughly studied and discussed within our team, we still eagerly need professional suggestions and feedback from scholars.

Thus, when we were informed that Prof. Kalvakolanu would conduct a lecture in our school that was primarily concerned with STAT3, a factor highly related to increasing tumor cell viability and promoting tumor cell proliferation, we sought his advice.

To conclude, Prof.Kalvakolanu notably illustrated how STAT3 considerably affects tumor growth and tissue invasion. While he did mention the loss of the cellular inhibitor GRIM-19 elevates STAT3 protein expression as well, it cannot fit well in our project, and thus, he recommended we make use of the downregulation mechanism of cellular genes using RNAi, which we regarded perfectly relevant to our preliminary experimental work and project design. Furthermore, Prof. Kalvakolanu stated that one prime advantage of our experiment is that bacterially delivered shRNAs can strongly inhibit tumor growth, making inserting shSTAT3 a preferable choice for our team.

The meeting with Prof. Kalvakolanu helped us to understand the best option for genes to apply in the RNAi mechanism. After much literature review, our team discussed the potential benefits and drawbacks multiple times, concluding that adding shSTAT3 in our plasmid is essential and that practical operation is available in the university lab.

As our previous literature study showed that STAT3 could enhance the function of PD-L1, we were then aiming to add shPD-L1 in the plasmid as well. Still, our team was unsure if designing one recombinant plasmid containing shSTAT3 and shPD-L1 sequences would be better or using two separate plasmids to insert, respectively. Therefore, our next step is to delve deeper into this topic by performing extensive literature studies and counseling.

Owing to our undecided choice of plasmid, we researched previous iGEM teams to see if similar issues occurred. However, our wish to draw on their experience was disappointed. Though a follow-up study on literature provided helpful information, we were still keen to obtain insights on this subject from our PIs.

Generally, our PIs clarified that designing only one plasmid containing shSTAT3 and shPD-L1 would be comparatively more desirable, silencing the two simultaneously. As our PIs explained, expressing multiple genes on a single plasmid can shorten research time and improve the accuracy and efficiency of expression. When there are two marker genes on the plasmid, the target gene can be inserted into one of the marker genes, which means that the recombinant plasmid only contains one marker gene, while the regular plasmid contains two marker genes.

The receptor cells without introducing plasmids do not have traits controlled by marker genes, the receptor cells with introducing ordinary plasmids have characteristics controlled by two marker genes, and the receptor cells with introducing recombinant plasmids only have traits controlled by one marker gene. This can accurately screen receptor cells containing recombinant plasmids based on the characteristics possessed by marker genes. In summary, expressing two genes simultaneously makes research more effective, precise, and efficient and saves research time and costs. Therefore, after a second article research and discussion, we believe creating a shSTAT3/shPD-L1 plasmid is plausible to silence both genes in one plasmid.

After resolving the eventual adoption of plasmid, our next step is to further validate if it is still essential to introduce suicide genes since our bacteria strain had altered to the delayed lysis strain χ11802.

Based on the preceding survey and enhancement of bacteria strain, we could ensure biosafety without introducing suicide genes. To assess the necessity of suicide genes, we aimed to meet with Prof. Ying Xin to gain input regarding a straightforward experimental design.

Prof. Ying Xin agreed with our concern that introducing suicide genes may not be necessary. She emphasized that χ11802 is a delayed lysis strain in which the essential genes asdA and murA for synthesizing the peptidoglycan layer in Salmonella are placed under the control of the arabinose promoter araCPBAD. The outstanding privilege of χ11802 lies in that it prevents the strain from synthesizing critical components of the cell wall in an environment lacking arabinose in the host, leading to the self-lysis of the cells and the release of plasmids. Accordingly, she agreed that as bacterial strain χ11802 can ensure biosafety, introducing suicide genes would be needless.

After the meeting with Prof. Ying Xin, we carefully scrutinized the possible effects of employing χ11802 without suicide genes by studying the literature in-depth. Combining all the information we received, we critically reflected on our previous work and decided not to introduce the suicide genes.

Our next step is to further discuss with iGEMers countrywide if the defects mentioned above in atomization inhalation possess a decisive influence on our project concerning lung cancer, as CCiC is coming up.

As we had been informed earlier, using aerosol inhalation to deliver bacteria may not be practical, and its viability lacked literature support. By then, we were right about conducting a presentation at the Conference of China iGEMer Community (CCiC). Therefore, we sought assistance from other iGEMers and the CCiC Committee to see if we could make aerosol-delivered bacteria therapy a reality or should specifically focus our project on colorectal cancer treatment.

During the conference of CCiC, we first presented our work on lung cancer to Chinese iGEMers online, with our leader discussing offline in the main venue in Hainan with another team on the therapeutics track, who were enthusiastic about our therapy. Though few teams are encountering such problems involving medicine administration, they were still concerned about our choice of aerosol inhalation. They suggested we search for additional information about whether aerosol can carry bacteria. Later, we met with Prof. Yuhan, a member of iGEM Foundation Safety Comment, to consult for professional feedback.

Prof. Yuhan Bao was hesitant about the topic of aerosol inhalation. He stated that barely any evidence might prove that aerosol could carry macromolecule or even Salmonella in our project. Essentially, we must adopt the most appropriate approach to medicine. It would be achievable to simply administrate our carrier with oral capsules for colorectal cancer. That being the case, we decided to concentrate our project on colorectal cancer treatment based on much discussion.

Our next step is to conduct a deeper investigation into the clinical treatment of colorectal cancer. For this, we reckon that it would be fundamental to reason our plan from a clinical point of view and that it is of great value to interview doctors of gastrointestinal surgery to improve the project.

After communicating with Prof. Dhan V. Kalvakolanu and Prof. Ying Xin, we returned to the realization of the characteristics of Salmonella itself as an intestinal parasitic bacterium. Then we went to the Gastrointestinal Surgery Department of China-Japan Union Hospital of Jilin University for a new round of Workshop, intending to find clinical recommendations that would benefit us. The purpose of this meeting with Dr. Yongchao Li from the China-Japan Union Hospital was to get feedback on our concept idea from a clinical point of view and to obtain his opinion on the relevance of our target cancer and its potential to impact healthcare.

Dr. Yongchao Li was fascinated by the concept of our project. Currently, China's diagnosis and treatment guidelines have never mentioned bacterial therapy as a way to treat tumors. As a clinician, through our description, he believes that our treatment method, whether used as a preventive treatment or as an essential part of combined therapy, is exceptionally pioneering. However, he still had some concerns about our chosen disease. Although Salmonella is an intestinal parasite, its invasiveness remains challenging to control. This way, even if the patient's tumor is eliminated during treatment, our bacterium is a delayed lytic bacterium.

However, the intrusion link is still unavoidable, and the side effects caused by this are undesirable. Therefore, Yongchao Li suggested we pay attention to colorectal cancer and make specific suggestions on our drug delivery methods. Colorectal cancer is a typical cancer death case in clinical departments and is closely related to daily eating habits. Therefore, if our idea can be used as a preventive drug, it may reduce the incidence of colorectal cancer while improving patients' happiness.

To make our drug design more perfect, we should explore cancers more relevant to the engineered bacteria we design and more relevant to human life, such as colorectal cancer.

To do this successfully, Dr. Li suggested we consult WHO data and relevant data analysis literature in China and talk to more hospitals. We have also successfully met with doctors from Jilin University Bethune First Hospital and Jilin University Second Hospital. After that, we further improved our ideas and tried to simultaneously promote our drug design to public health and clinical treatment.

After deciding to research colorectal cancer, we further focused on some points requiring additional attention in implementing the treatments we hope to advance drug delivery systems and cost control. To this end, we went to Huakang Pharmaceutical for a seminar to understand the difficulties and obstacles in realizing what we want to do in the entire industry chain. Chuangui Liu, one of the leaders of Huakang Pharmaceuticals, discussed with us, and we also discussed helping developing and developed countries to enjoy the medical benefits of innovative drugs simultaneously.

Chuangui Liu is very interested in the innovative drugs we are working on. Regarding the current method of drug delivery, Manager Liu said there are clear classifications and regulations in the Chinese Pharmacopoeia, and it is recommended that we make capsule pharmaceuticals under the guidance of the Chinese Pharmacopoeia. At the same time, we also consulted on the feasibility of granular drugs, water-soluble drugs, and injectable products. Still, Manager Liu expressed doubts about this, believing that drugs and water-soluble drugs will be decomposed by gastric acid in the stomach, which will reduce drug efficacy.

To ensure the stable effectiveness of the drug, safety problems arise due to excessive dosage of the drug, which leads to exaggerated immune response; at the same time, there are significant concerns about the biological safety of injectable products. Therefore, Manager Liu suggested that we pay attention to capsule products that can be maintained in the colorectum and continue to understand the technical links required in the development process of oral drugs, including pharmacodynamic research, ADME research, safety assessment, bioequivalence, etc.

To further confirm the biosafety of our project, we need to inquire and discuss relevant international organizations and domestic regulations on bacterial biological products to help us be more credible and feasible in biosafety.

To demonstrate the feasibility of biosafety, we went to the Jilin Provincial Center for Disease Control and Prevention and studied relevant regulations. We found that the international openness to bacterial biological products is currently greater than domestic regulations. At the same time, we also found that domestic laws are gradually opening up.

After previous discussions, our team conducted a series of meetings. It was thought that liposomes might be used for drug delivery. Since PI. Yang is an expert in molecular biology, we approached him this time and hoped he could make professional suggestions on the plasmid delivery system. Propose a feasible solution for the implementation of our RNAi technology.

Our communication with government organizations is also a new experience as a team of undergraduates. We discuss how Salmonella, as the causative organism of foodborne illness, can be controlled under the Centers for Disease Control. At the same time, we and Director Sun Bingxin learned about the current national approval strains for bacterial biological products and interpreted the relevant regulations.

We learned through discussion with clinicians that our current experimental design is insufficient to reflect the human immune environment and, therefore, has significant limitations and unpredictability in clinical use. So, we planned to interview professionals to seek a better approach.

To recognize the shortcomings of our current project and to improve our experiment design in terms of biosafety, JLU-NBBMS invited Prof. Carlo Vittorio Cannistraci from the Laboratory of Brain and Intelligence, Tsinghua University, to be interviewed to discuss the design of our current project and how to avoid the limitations and drawbacks of cellular experiments under the constraints of the current rules of iGEM competition.

First of all, we briefly introduced our project design to Prof. Cannistraci, who then put forward some of the existing research results in predicting the development of diseases and therapeutic efficacy in the context of our project, also mentioning the combination of intelligence and network science approaches as he recommended us experts in this direction. Since the iGEM competition has been mainly concerned with the issue of experimental animals in the last two years, animal experiments should be avoided as much as possible, except in cases where their use is mandatory.

Prof. Cannstraci introduced us to organoids, which refer to tissue analogs with a particular spatial structure formed using adult or pluripotent stem cells in three-dimensional culture in vitro. Although organoids are not human organs in the true sense of the word, they can mimic real organs in terms of structure and function. They can mimic the structure and function of in vivo tissues to the greatest extent possible and can be stably cultured for an extended period. It has enlightened us on the limitations of cellular experiments that had plagued us for a long time, and we have benefited from Prof. Cannstraci's interview, which has given us a new direction to improve our project and optimize our experiments.

Based on the valuable information he brought, we are considering using organoids in future experiments to better simulate the human immune environment for a more rigorous experimental design and to better achieve expected results.

Expanding on our discussions with personnel from the Disease Control Center and clinical physicians regarding our pharmaceutical research proposal, we sought guidance from our medical-legal instructor to enhance the feasibility of our project. This interview aimed to acquire a deeper professional understanding of the relevant laws and regulations governing secure pharmaceutical manufacturing and drug marketing. This, in turn, serves as a guiding compass for the future development of our project.

During our discussions, we gained insight into China's primary laws and regulations concerning drug research and development for market approval, such as the 'Drug Administration Law of the PRC' and the 'Provisions for Drug Registration.' These legislative measures meticulously regulate various aspects of drug research, production, sales, and usage to ensure the safety and efficacy of pharmaceuticals. Additionally, we highlighted the implementation of the Marketing Authorization Holder system for drug approval in China. This system allows drug research institutions and pharmaceutical companies with advanced technologies to apply for drug marketing authorization in the holder's name, obtain a drug registration certificate, and introduce the product to the market while bearing responsibility for the entire product life cycle. This necessitates that our partners, including medical laboratories or consultants, meet specific qualifications and possess the necessary authorizations.

In conclusion, our dialogue underscored the importance of safety for drug research institutions and manufacturing companies. According to the law, post-market safety monitoring is mandatory to track a drug's safety throughout its entire product life cycle.

Following consultations with medical and legal professionals, our team has comprehensively understood the rigorous legal framework surrounding drug research and market approval. We rigorously adhere to these laws and recommendations throughout every project phase. Our next step involves interviewing specialized pharmaceutical research firms, such as Changchun Boxun Biotechnology Co., Ltd., to gain insights into pharmaceutical production.

The company is a high-tech biotechnology enterprise specializing in in vitro diagnostic test reagents. Building on our in-depth understanding of the legal regulations governing drug research and market approval, our goal is to refine our knowledge of the production process for research products transitioning from the laboratory to the market through collaboration with this biotechnology company. This collaboration will promote increased interaction and synergy between academia and industry, further guiding the progress and translation of our research project.

The company's personnel led our team on a tour of the company's research and development department and laboratories, resulting in extensive discussions. The R&D department manager elucidated the company's core business, product lines, and key technical features. The company specializes in researching and producing in vitro diagnostic test reagents. It has achieved notable success in recent years, particularly in micro-column gel immunodetection technology, aqueous gel immunoassay technology, and monoclonal antibody technology. During the interview, the R&D department manager emphasized that drug production is a complex and precision-demanding process requiring high specialization and strict quality control.

Our team presented the current status of our research project, progress in our experiments, and our plans. The company's technical staff underscored that the industrialization of research outcomes, involving the transformation of research achievements into marketable products, encompasses a broad spectrum. The company's personnel commended our approach after learning that we had previously sought guidance from legal professionals to study the relevant laws and regulations. It provided us with a comprehensive explanation of the standards to be followed in various domains, including clinical trials, quality control, raw material procurement, formulation production, packaging, and storage, all within the framework of legal regulations. Furthermore, the company's personnel particularly emphasized the importance of closely monitoring the pharmaceutical lifecycle and dosages of the project, aspects we should prioritize in subsequent phases.

Following the guidance provided by Bioxun, we have now gained a clear understanding of the procedures and standards underpinning pharmaceutical industrialization, which serves as a significant guide for the advancement of our project. Additionally, the company's personnel recommended that we delve into issues related to intellectual property in the drug research and production process to enhance patent protection for our project. As a result, our next step involves seeking advice from Professor Wang Fang to acquire relevant knowledge concerning intellectual property and patent protection.

So far, we have completed discussions with clinicians, law professors, pharmaceutical companies, and many others about the business of the drugs we have developed. To further explore this project's future feasibility and development, we have approached Professor Wang Fang of the College of Basic Medical Sciences of Jilin University to give us more professional advice. We expect him to bring us a more comprehensive perspective from drug R&D to translating results.

Prof. Fang Wang suggested that a long cycle, high risk, and high return characterize innovative drug research methods. On the road to new drug research and development, we face many challenges, among which we need layers of defense and barriers to intellectual property rights in the whole cycle. There is a common saying in the pharmaceutical field that "without patents, there are no new drugs." From the perspective of patents, patent protection usually refers to the protection of products or methods; pharmaceutical patents can also be categorized into product and method patents. Intellectual property management of drugs runs through the whole process of research, development, and operation.

From project screening, drug discovery, and structure optimization to clinical development and drug registration, market competition in all intellectual property rights issues needs to be addressed. The significance of pharmaceutical patents lies in stimulating innovation, encouraging investment, competition protection, accelerating technology transfer, and maintaining intellectual property rights. In conclusion, patent protection in the pharmaceutical industry is a complex and vital issue, and obtaining high-value and high-quality patents is essential for drug development. We aim to further refine the strain to file a patent application early.

After consulting with Prof. Wang Fang, our team has gained a new understanding of translating the results. Our next goal is to optimize the project's safety and feasibility and continue to work hard for the transformation into a finished drug. At the same time, we have actively reviewed the relevant intellectual property laws and regulations in anticipation of an early patent application. Next, we intend to go to relevant companies to seek knowledge about drug effectiveness to further improve the project.

After many aspects of discussion, we have made a series of improvements and refinements to our project. To advance the finished drug development more smoothly to benefit more groups in the future, we went to WISH TECH, hoping to get help from the enterprise side.

Cong Cong, who is familiar with experimental techniques, pointed out that plasmids usually have excellent stability and are not sensitive to temperature. Viruses are different. They generally need to be stored at low temperatures and are very sensitive to temperature. Repeated freezing and thawing also have a significant impact on viability. Moreover, plasmid vectors have a large carrying capacity, are easy to operate, and are relatively cheap. We aim to apply new technologies to benefit more people, so universality and economy are essential. Therefore, plasmids are undoubtedly our best choice. The PI also explained that chemically synthesized siRNA has a short half-life in the body, is easily degraded, and needs to be administered continuously during treatment.

The shRNA expression system can self-replicate in tumor cells and continually express shRNA-silencing genes. The PIs suggested that we can choose to construct shRNA plasmids to conduct our research. Cong Cong, who is familiar with experimental techniques, pointed out that plasmids usually have excellent stability and are not sensitive to temperature. Viruses are different. They generally need to be stored at low temperatures and are very sensitive to temperature. Repeated freezing and thawing also have a significant impact on viability.

Moreover, plasmid vectors have a large carrying capacity, are easy to operate, and are relatively cheap. We aim to apply new technologies to benefit more people, so universality and economy are fundamental. Therefore, plasmids are undoubtedly our best choice. The PI also explained that chemically synthesized siRNA has a short half-life in the body, is easily degraded, and needs to be administered continuously during treatment.

The shRNA expression system can self-replicate in tumor cells and constantly express shRNA-silencing genes. The instructor suggested that we can choose to construct shRNA plasmids to conduct our research.

After professional guidance, we finally constructed a shRNA plasmid to silence oncogene expression. Initially, we chose the Therapeutics, looking forward to using this synthetic biology approach to treat cancer. Next, we will conduct in-depth research on how to deliver plasmids into the body to achieve a highly innovative and rational method and determine the use of plasmids as vectors rather than viruses. The project track has been initially chosen as the Therapeutics!

Building upon discussions and consultations with the company, our team, considering the need to ensure the feasibility and safety of the project, has decided to focus our efforts on reducing drug side effects. We sought advice from Prof. Ming Yang, a professor at the School of Basic Medical Sciences, Jilin University, on drug side effects. We look forward to his expertise in providing professional recommendations and improvement measures.

Prof. Ming Yang defined drug side effects as "evidently harmful or unpleasant reactions caused by interventions related to drug usage." They predict future harm from drug administration and ensure prevention, specific treatment, drug regimen alteration, or product discontinuation. Drug side effects are categorized into six types: dose-related, non-dose-related, time-related, dose and time-related, discontinuation, and treatment failure. The reasons for side effects occurring in only some individuals may be related to variations in drug metabolism and the production of active metabolites.

However, the mechanisms of individual response variations are often unknown. Susceptible factors can be intrinsic (such as genetic polymorphisms, age, gender, race) or acquired (such as drug interactions, diseases, liver and kidney dysfunction). Some side effects make the drug an absolute contraindication for future use, while in other cases, the medication may be cautiously reevaluated. If a specific drug produces side effects, other drugs with similar pharmacological effects may have the same effect and are generally best avoided.

After consulting with Prof. Ming Yang, our team has gained a more professional understanding of side effects. In the next step, we will optimize the safety of the project. We are actively researching relevant professional information on drug side effects to achieve more profound optimization of the existing foundation. Furthermore, we plan to delve into the patient population to explore their acceptance of the new drug.

Following discussions with Professor Yang Ming regarding drug side effects, our team has shifted its focus to the patient level. Through various modifications to the drug, we aim to understand the extent to which patients accept the new drug and their opinions on our research.

We conducted a patient survey, ensuring the complete security of patients' basic information and the inclusivity of various demographic groups. The survey included questions about patients' basic lifestyle habits, sources of obtaining medical information, their level of awareness about cancer, their opinions on bacterial therapy, and their acceptance and expectations of the new drug. Through our promotional efforts, we collected a sufficient sample of data and generated meaningful results that aligned with our study design. Using patient feedback, we will further optimize our project to produce the most effective drug within the limits of patient acceptance.

We have gained a deep understanding of patient's concerns and expectations regarding the drug through precise data collection and calculations. We have worked diligently to optimize our drug by integrating practical implementation with patient needs. In the next step, we plan to have detailed discussions with a biotechnology company regarding the issues surrounding clinical trials for the drug.

Through continuous optimization, our drug research has reached a new level. We are no longer satisfied with its use solely within the laboratory; our ultimate goal in exploring this new therapy is to bring it to market as a pharmaceutical. Merely demonstrating effectiveness in in vitro and in vivo experiments is insufficient; clinical trials are the crucial step in determining its utility.

The professionals from Qizhong Company led our team members on a tour of their company and engaged in detailed discussions with us. After learning about our project, the company personnel expressed great interest in the potential of our project to treat cancer. Considering our project's ultimate goal is to bring it to market as a pharmaceutical, the company personnel provided advice on biosafety and drug administration pathways. They educated us on biosafety laws and regulations. While the country supports the development of biotechnology, it also mandates the necessity of risk assessments to trace critical factors, ensuring no harm to biosafety.

Regarding drug administration pathways, the company personnel suggested an oral capsule method different from our team's previous inhalation approach. Capsule drugs are more accessible to take than other medications and dissolve more readily in the digestive tract, facilitating better absorption. Capsule drugs also favor targeted release, making them more efficient in targeting cells and reducing irritation to other parts of the body.

Following discussions with professionals, we have preliminarily decided to use capsules for our medication. Our team has gained a deeper understanding of the significance of biosafety in drug development. In the upcoming phases, we will explore various aspects of biosafety with more organizations and enhance our capabilities to ensure safe pharmaceutical practices in all areas.

Regarding biosafety, we constantly ensure that the content of our research meets the requirements of the iGEM competition and that no biosafety issues will arise due to breakdown. This is a principle that we as a team have always adhered to from beginning to end. Before the overall wet experiment begins, we have already arranged the timetable for experimental safety training and signed a practical safety commitment letter for group members with Sun Wei, the person in charge of biosafety at the college. Sun Wei, an expert in biosafety at the College of Basic Medicine Science of Jilin University, discussed with us the preparations that a newcomer needs to make when entering the field of scientific research, as well as the aspects that need to be paid attention to in terms of biosafety.

Wei Sun explained to us the aspects that need attention during the entire experimental process, from laboratory to cell-to-cell safety factors. We are also introducing the experimental strains we study with Dean Sun. Dean Sun said that what we have done is groundbreaking, but we cannot ignore the pathogenicity and invasiveness of Salmonella itself as a pathogenic bacterium. Since our team hopes to apply it to the treatment process, we must Be raised as to whether the spread of bacteria could arise as a result and the line between bacteria as biological agents and biological weapons.

After conducting theoretical and basic safety training in the school, our PI took us to Changchun Saidi Biotechnology Co., Ltd. for biosafety training and introduced us to the pharmaceutical process of biological products while understanding it. International principles for different levels of biosafety.

Our team's focus and implementation on biosafety is unquestionable. We have always adhered to the requirements of iGEM and the biosafety principles stipulated by international organizations and constantly verified experimental verification and biosafety during the research process. More related to Safety: Click to jump to the Safety interface.

On the iGEM biological form, we not only communicated with Dr. Sun Wei but also hoped to obtain recognition from relevant government departments again. To this end, we went to the Jilin Provincial Center for Disease Control and Prevention based on the safety form and the applicable regulations on recombinant engineered bacterial biological products. Communicated with all parties and achieved satisfactory results.

We learned that during our research, national policies were also changing. The regulations "Management and Quality Control of Bacteria and Viruses Used for Production and Testing of Biological Products" were proposed, providing legal basis support for engineered bacterial biological products in China and allowing We have seen the domestic vision of promoting research in developing countries to keep up with the progress of developed countries.

Among them, there are the following principles and standards for the verification of main seed batches of bacterial strains for the production of recombinant engineering bacteria: generally should include culture characteristics, colony morphology, and size, Gram Microscopy using other staining methods, resistance to antibiotics, biochemical reactions, culture purity, complete gene sequence determination, target product expression, transmission electron microscopy, target gene sequence determination, identification of foreign genes and host genes, foreign genes Integration into host chromosome detection, exogenous gene copy number detection, integrated gene stability test, identification of target products, plasmid enzyme digestion map, and other projects.

We discussed with the Jilin Provincial Center for Disease Control and Prevention the feedback from iGEM officials on our safety form and determined that we would add settings. In the worst case, if our bacteria spread into nature, the engineered bacteria we designed will also not cause a wide range of infection problems and ensure overall safety. Please see the protection for further description.

Finally, as our project nears its conclusion, we had the privilege of meeting with Prof. Xinmin Li. Prof. Xinmin Li has dedicated many years to talent development, academic exchange, and international medical education. We hope that he can provide us with more professional insights and recommendations for the future of our project from a higher perspective.

One primary theme mentioned in our session with Academician Xinmin Li is the critical impact of science and technology on the health environment of human society. More specifically, he pointed to the fact that synthetic biology has great potential in developing countries to ensure equal medical treatment for more people around the globe.

Academician Xinmin Li points out that this may sound very idealistic, but it is not impossible to achieve. Synthetic biology can be applied in various channels. For example, the transformation project we are doing to treat diseases has excellent potential to improve the medical environment in poor areas and avoid problems such as medical treatment, hospitalization, and insufficient funds. At the same time, the role of synthetic biology in the world's public health should not be underestimated, and global public health problems have attracted more and more attention in recent years. The ideal future is for anyone to enjoy the same medical rights everywhere, rich or poor.

Our project will help to cope with the severe challenges in the treatment of malignant tumors, bring new hope and infinite possibilities for tumor treatment to achieve efficient, low-toxic bacterial therapy in the application of tumor treatment, break tumor lines, let patients no longer need to bear the pain of surgery and therapy, significantly improve the patient's quality of life, allow life to continue. Our project can not only provide new weapons and good news for cancer patients but also reduce the medical burden on families and society.