In our project, we ingeniously utilized the Salmonella delivery system and designed a unique toxin-antitoxin system to prevent plasmid loss, significantly enhancing safety. Additionally, in collaboration with Guangxi-U-China, JLU-NBBMS, PekingHSC, Peking, and Tsinghua, we have completed the reference manual for microbial-mediated tumor therapy in synthetic biology. Our aim is to offer a practical and meaningful solution to the common challenges faced by iGEM teams dedicated to conducting projects on tumor microbial-mediated therapy in the future, including safety, ethics, and engineering design. Our work has the potential to provide novel insights, raise awareness about customized bacterial delivery methods, propose innovative strategies for reducing plasmid loss, and advance the field of cancer therapy.

Tumor starvation therapy entails inhibiting tumor growth by obstructing its nutrient supply. Unlike normal cells, cancer cells require more abundant nutrients and energy to sustain abnormally rapid proliferation. This indicates that tumor cells are more responsive to intracellular glucose levels. In comparison to conventional tumor starvation treatments, our project introduces an innovative approach by utilizing engineered bacteria to deliver glucose oxidase (GOx). When these engineered bacteria infect tumor cells, they can inject GOx into the cells through the T3SS system. This system catalyzes the oxidation reaction of glucose, producing gluconic acid and hydrogen peroxide (H₂O₂) ¹, thereby cutting off the nutritional source of the tumor cells and achieving the purpose of starvation treatment.

Chemo-dynamic Therapy (CDT) is a type of tumor treatment technology that relies on the transformation reaction of endogenous chemical products. It utilizes the tumor microenvironment to activate the Fenton reaction and generate powerful oxidative hydroxyl radicals (·OH) to eliminate tumor cells. Building upon the aforementioned tumor starvation treatment, we employ S. typhimurium to deliver GOx to tumor cells. GOx catalyzes the oxidation reaction of glucose, resulting in the production of H₂O₂. Subsequently, hydroxyl radicals (·OH) and reactive oxygen species (ROS) are generated through the redox-active Fe²⁺ catalytic process, ultimately triggering the Fenton reaction. This approach is referred to as chemical kinetic therapy, inducing tumor cell ferroptosis.

In conclusion, in this module, we utilized S. typhimurium engineered bacteria to simultaneously facilitate tumor cell therapy and chemical therapy. This novel approach presents a new concept for future cancer therapy.

For more details, refer to Results: Fenton.

Silencing specific genes through RNA interference (RNAi) is an effective method for reducing gene expression. However, achieving efficient delivery of small interfering RNAs (siRNAs) remains a concern. Most delivery strategies have focused on development of chemically modified or biologically coupled siRNAs. With the increasing attention given to microbial-based cancer therapy, the utilization of bacteria for delivering substances into cells presents a significant advantage. Bacteria-mediated RNAi is an effective combination of bacterial therapy and RNAi therapy².

Cross-kingdom RNAi is a conserved mechanism in many species that can occur in host-symbiotic microbial interactions. Gene silencing in mammalian cells can be achieved with the help of bacteria carrying specific plasmids and cross-kingdom RNAi mechanisms. Here, our team applied engineered Salmonella to mediate RNAi in the iGEM project for the first time, silencing SLC7A11, which is one of the most critical upstream regulators of ferroptosis. We further validated the feasibility of engineered bacteria-mediated RNAi, providing a new idea for engineered bacteria to treat tumor therapy.

Our engineered bacteria can produce large amounts of shRNA and release it into the cytoplasm of the host cell. The system is based on a plasmid. This plasmid we constructed contains the HlyA gene and the T7 RNA polymerase gene. The HlyA gene codes for Listerolysin-O³, which helps release genetic material from the vesicles, greatly improving the efficiency of gene silencing. The oligonucleotide encoding the shRNA of SLC7A11 was inserted into the cloning site of the plasmid. By turning this constructed plasmid into attenuated Salmonella typhimurium, we can obtain a strain that will mediate RNAi and silence SLC7A11.

For more details, refer to Results: shRNA.

Two functional plasmids are used in many iGEM projects, which often face a more severe plasmid loss, and our project is no exception. Therefore, we designed a unique "logical suicide circuit" to maintain both plasmids, and at the same time, we are able to induce suicide in the engineered bacteria through this logical circuit. Toxin-antitoxin systems are often used to maintain plasmid loss and also to regulate the suicide of engineered bacteria. Faced with the problem of plasmid maintenance, a common way is to insert the toxin into the helper plasmid and the antitoxin into the functional plasmid, and when the functional plasmid is lost, the engineered bacteria are killed by the toxin. However, this approach will fail in the face of two functional plasmids, and this approach cannot maintain the helper plasmid, so that cannot maintain the functional plasmids for a long time.

Our devised "logical suicide circuit" overcomes this problem. The engineered bacteria are killed regardless of which plasmid is lost. At the same time, this circuit also has the function of artificially inducing the suicide of the engineered bacteria. We realized two functions of simultaneously maintaining plasmids and inducing engineered bacteria suicide through the toxin-antitoxin system, providing a safe and effective means for future iGEM teams to apply engineered bacteria to treat human diseases.

For more details, refer to Results: Safety.

The 1^st iGEM Greater Bay Area Synthetic Biology Industry-Academia-Research Forum (iGBA), hosted by BNUZH-China, UM-Macau, HKUST and SZU-China, promises to be a groundbreaking event filled with uniqueness and innovation (For more details: Integrated Human Practices). We firmly believe that this forum will serve as a beacon of inspiration for future iGEM teams planning workshops. Our contributions are manifested in the following specific aspects.

The first aspect revolved around the expansion of conference themes. In the traditional iGEM conferences, the scope of discussion was often limited to the communications between iGEM teams. However, iGBA has taken a progressive approach by broadening the topics to encompass collaborative efforts between industry, academia, and research institutions. By bringing together iGEM teams, representatives from companies and research institutes, iGBA delved into academic discussions on diverse subjects across the entire value chain. These included product development strategies, research outcome commercialization, and the overall direction of industry development.

The second aspect revolved around the diversification of participant roles. In addition to iGEM teams, iGBA extended invitations to representatives from various biopharmaceutical companies and research institutes. This created a valuable platform for meaningful face-to-face interactions. Notably, the diversity of participants also encompassed individuals from the Greater Bay Area. We envision iGBA as a catalyst for fostering enhanced communication and collaboration among iGEM teams, industry experts, and academic institutions within the Greater Bay Area.

The third aspect revolved around the innovation in the format of the conference. Diverging from the conventional practice of iGEM teams taking turns to showcase their projects on stage, iGBA had creatively introduced a format emphasizing expert lectures and field visits to company facilities, supplemented by interactive booths. This approach provided participants with a firsthand experience of the practical applications and challenges linked to research outcomes, thus facilitating the integration of theory and practice.

In the Integrated Human Practices part, we incorporated the theoretical principles of engineering to establish the framework for our endeavors. Through an engineering lens, a project's life cycle typically encompasses phases such as planning, design and development, acceptance and maintenance management. In the context of an iGEM project, we segmented our undertaking into preparation, exploration and industrialization stages. Consequently, our integrated human practices involved conducting targeted activities during each phase, guided by this approach, and promptly incorporating the acquired insights into the overarching project design.

In the Education part, we employed the theory of communication to guide our activities. From a communication standpoint, we understood that audiences have an innate curiosity for diverse and engaging media formats. It is within this context that the infusion of art elements offers a novel and captivating medium for information dissemination. Art forms such as music, film, and theater can present scientific concepts and principles in a more intuitive and emotional way, and achieve an emotional resonance with the audience. Inspired by this realization, BNUZH-China had crafted the "Art Alive" series as an integral part of our education framework (For more details: Education). This innovative approach sought to unravel the intricacies of science by intertwining it with the expressive power of art. Through this creative fusion, scientific knowledge comes alive in vibrant, mesmerizing ways.

Compared to other fields, synthetic biology is currently considered a more specialized field with a limited audience. We noticed that purely disseminating knowledge about synthetic biology can be somewhat bland and typically attract only biology students and professionals in the field.

In light of this, BNUZH-China spent considerable time and effort this year brainstorming ways to broaden the reach and impacts of our education activities. After much deliberation, we stumbled upon a remarkably effective approach: interdisciplinary collaboration. Over the course of nearly one year, we forged partnerships with various student organizations and clubs such as the Guitar Club, Drama Club, Debate Team, Programming Club, and an academic organization affiliated with our college (For more details: Education).

This ingenious strategy not only breathed fresh air into our educational initiatives but also captured the attention of individuals from diverse realms, including music, drama, debate, programming and so on. The expanded audience was intrigued by the world of iGEM, synthetic biology and our project.

We couldn't be prouder of the fact that our collaborative events have received resounding acclaim from our partner organizations. The Debate Team, for instance, expressed their impressions, saying, "The synergy we achieved through our collaboration with iGEM was truly marvelous! It left us inspired, prompting us to explore more interdisciplinary approaches for our own activities. By any chance, could you share your event planning blueprint with us?"

Hence, we strongly believe that this experience holds immense value as a reference and inspiration for future iGEM teams embarking on education activities. May they recognize the transformative potential of breaking free from disciplinary silos, forging unconventional partnerships, and embracing the power of multidimensional education. Together, we can carve a path toward a more inclusive and dynamic scientific landscape.

Through discussion and exchanges with our partnering teams JLU-NBBMS and Peking this year (For more details: Collaboration), we have found that many issues are unavoidable in the design and conceptualization process of microbial-mediated tumor therapy. For example, how to handle ethical concerns caused by engineered bacteria? How to address safety issues in tumor treatments? How to ensure engineered bacteria can specifically colonize tumor areas and minimize damage to normal tissue cells?

These issues are common problems encountered by teams working on microbial-mediated tumor therapy. They are relevant not only to our project about engineered bacterial-mediated ferroptosis in tumor cells this year but also to the design of more extensive engineered microbial chassis in tumor treatment projects. To discuss these issues together, we have collaborated with JLU-NBBMS and Peking to establish the Microbial Cancer Therapy Discussion Group in China on May 8th of this year. After the 10^th Conference of China iGEMers community, our seminar group attractedmore iGEM teams, including Guangxi-U-China, PekingHSC, Tsinghua, USTC, CPU-China, Jilin-China to join in. During the First Meeting of the iGEM Cancer Biotherapy Congress in China on July 27^th this year (For more details: Collaboration), we proposed to compile the “Microbial Mediated Tumor Therapy in Synthetic Biology” reference manual. We hope to provide practical and meaningful solutions to the typical difficulties faced by safe, ethical, engineered design to the iGEM team and other scientific research teams aiming to conduct microbial therapy oncology projects in the future.

We clarified the framework, content, and division of labor for the manual among the participating teams. Subsequently, we embarked on a lengthy writing process, continuously optimizing the details of the manual until a final draft was completed at the end of September this year.

When writing this manual, we sought the input of numerous former iGEM team members and advisors. Our intent was to create a manual that can be utilized by any future team in need and easily integrated into their unique team designs. The content of our manual encompassed various aspects including background information on biotherapy in cancer treatment, engineered chassis design, ethical considerations for biotherapy, and human practices. Throughout the writing process, the collaborative efforts of the six teams fostered an exceptionally close relationship. We extensively reviewed previous iGEM projects and research papers pertaining to microbial therapy for tumors, engaging in comprehensive discussions on module design and project ethics on multiple occasions.

Our team spent a lot of time and energy from determining the corresponding framework to collecting the drafts of each team and finally sorting out and typesetting the design. We also listened to the opinions and suggestions of many participating teams, and revised the manual. Finally, at the end of September, representatives of each team completed their signature on the back of the manual. On the one hand, we hope that this handbook will provide solutions from multiple perspectives to many common problems encountered by future iGEM teams on related topics, and consider issues they may have overlooked. We also hope that the manual will provide a reference for other scientific researchers and will be further improved in the future. On the other hand, the manual is full of pictures and texts, and the content is vivid. We hope to apply the manual to the campus popularization of science in universities that more students who are just interested in biology and medicine or eager to learn more about cancer can benefit from it.