Overview

We have modified the attenuated Salmonella in a novel way, offering a fresh perspective on drug delivery. Additionally, we have preliminarily established an efficacy scoring model tailored for "engineered bacteria", aiming to provide a dynamic efficacy analysis model for future iGEM teams using bacteria as chassis. Furthermore, as advocates, we have summoned 18 teams to collaborate on a Biosafety & Ethics White Paper. Our goal is to offer practical and meaningful solutions to the shared challenges faced by iGEM teams in pursuing future projects related to microbiome-mediated tumor therapy, including safety, ethics and engineering design. Our work holds the promise of shedding new light on bacterial-based cancer therapy and presenting a novel strategy for drug delivery.

Laboratory

Novel modification methods for attenuated Salmonella

The chassis we used is the delayed lysis Salmonella strain χ11802 that has undergone deletions of the asd and murA genes, rendering the mutant bacteria incapable of synthesizing a complete cell wall. Meanwhile, we have inserted the murA and asd genes into the companion expression plasmid, but the expression of these genes is under the control of the araC pBAD promoter. This means that the murA and asd genes can only be expressed in the presence of arabinose. After the Salmonella enters the body, the arabinose concentration will gradually decrease, and the Salmonella will gradually autolyze. In this way, it further guarantees the safety of the attenuated Salmonella. Additionally, we have further knocked out the msbB gene. In Salmonella, the msbB gene encodes an enzyme called myristoyltransferase, which plays a crucial role in the biosynthesis of lipopolysaccharides. By knocking out this gene, we reduced the toxicity of the attenuated Salmonella.

Approach for Nanoparticles attached to Bacteria

Nanoparticles transform from "carriers" to "missiles". Nanoparticles are attached to the cell wall of attenuated Salmonella through amide bonds. Driven by Salmonella, which tends to migrate towards hypoxic environments, the nanoparticles are carried towards tumor cells, thereby reducing the toxic and side effects in other parts of the body caused by the accumulation of chemotherapy drugs administered through systemic injections.

To learn more about the novel modification methods for attenuated Salmonella and approach for Nanoparticles attached to Bacteria, see Design.

Efficacy Analysis Model

Our team has developed an efficacy analysis model tailored for "engineered bacteria", modularizing bacterial functions into "targeting modules", "functional modules" and "safety modules".

Firstly, the targeting module is divided into two types: "bacterial-chassis-based targeting" and "specific-binding-based targeting". For the former, we harness the inherent properties of bacteria and achieve targeting functions through genetic engineering. For instance, in this project, we utilize the hypoxic promoter pnirB as a targeting switch for our engineered bacteria in the tumor microenvironment. For the latter, we achieve specific targeting by expressing fusion proteins, such as single-chain variable fragments (scFvs), aptamers and specific proteins, on the bacterial surface.

We employ structural modeling (e.g., AlphaFold) and molecular docking (e.g., AutoDock Vina) to optimize the structure of fusion proteins, enhancing their stability. Simultaneously, through molecular dynamics simulations (e.g., Amber22), we improve the affinity and binding stability of fusion proteins, thereby enhancing targeting efficiency.

In terms of functional modules, the realization of all functions relies on gene expression. We emphasize the gating of the system, typically achieved through the regulation of promoters by external factors. We validate the feasibility of our approach through a combination of mathematical modeling and molecular docking. However, if the promoter used has already been applied in the past, whether additional validation is required depends on the specific situation. For instance, certain something that are effective in Escherichia coli may not function similarly in Salmonella.

The application of different mathematical methods depends on the functions we aim to achieve. For RNAi, we need to screen for drug resistance genes to be silenced. By analyzing the TCGA and GEO databases and using medication status as a label, we screened for drug resistance genes corresponding to different tumor types and drug usage scenarios. We conducted a prognostic analysis and ranked the drug resistance genes based on their risk coefficients, which served as candidate targets for siRNA silencing. In terms of safety, we mainly focus on two key factors: firstly, the post-injection impact of the engineered bacteria on the body's immune system; secondly, the safety verification of delayed lysis strains. Firstly, upon entering the human body, the engineered bacteria will be recognized by the immune system as foreign substances, potentially triggering an immune response. We evaluate the intensity of the immune response by analyzing the recognition mechanisms of the immune system towards the engineered bacteria. This involves analyzing the activity of immune cells such as macrophages and T cells, as well as monitoring the secretion levels of cytokines.

Secondly, in terms of the verification of delayed lysis strains, our concern is whether the engineered bacteria can effectively lyse and clear themselves after achieving their intended function in the target area, thereby avoiding adverse reactions caused by long-term retention. Delayed lysis is designed to achieve precise control by regulating the expression of specific genes or the initiation of metabolic pathways. In our dry-lab experiments, we use mathematical modeling to predict the lysis time and drug release curves of the engineered bacteria, ensuring that the timing of lysis meets therapeutic needs while avoiding excessive bacterial proliferation in the body.

To learn more about the Efficacy Analysis Model, see Model.

Part

JLU-NBBMS 2024 has designed and characterized a vast array of basic and composite components, which have been uploaded to Parts.

iHP & Education

Beyond the specific activities conducted, the significance lies in its comprehension and reflection on the iGEM concept and the needs of all stakeholder groups. All these deliberations culminate in the formation of an expansive iHP network, which in turn feeds back into the entire Education process, fostering a mutually beneficial two-way feedback loop between JLU-NBBMS and its stakeholders. None of our activities exist in isolation but rather vivid within a flexible and dynamic intellectual trajectory. Under each filter, we have meticulously documented the entire thought process, allowing iGEMers to discern the significance of each activity.

iHP Entrepreneurial Sector

We offer an exceptional resource for future iGEM teams that aspire to work in the therapeutic field. While iGEM projects often emphasize the scientific research process, realizing innovative potential in the market can be even more challenging. The JLU-NBBMS team has provided a comprehensive timeline for market research and formulated a detailed business guidebook. This is not only a valuable guide but also a source of inspiration for synthetic biology startups. We have visualized the path from the laboratory to commercial success, encouraging team members and other iGEMers to think beyond the laboratory. From background research to clinical trials, legal regulations and quality control throughout the process, to product launch strategies and promotion methods tailored to stakeholders with different levels of awareness and consumption power, our content enables other iGEM teams to have sufficient support in modeling commercialization strategies in the real world.

To learn more about entrepreneurship,please visit HP.

iHP Ethics Sector

Based on Silver HP's detailed analysis of stakeholders, we identified the four most concerned groups of interest in our project and discovered the intertwined relationships and ethical challenges among them. Recognizing the importance and complexity of ethics, we formulated a Biosafety & Ethics White Paper and expanded it into a holistic ethical narrative. Our discussions on animal ethics, medical ethics, clinical trial ethics, scientific and technological ethics, and social ethics continuously feed back into our own practices, fostering a cyclical process of recognizing the current situation, attempting to change it, and re-evaluating our understanding. The White Paper integrates insights from 18 teams, drawing on their own current situations and the safety and ethical considerations of previous outstanding teams. It includes track introductions, project descriptions, laboratory safety, safety switches, ethical regulations analysis and impressive case studies, providing a blueprint for nearly all villages to guide them through the entire process from track selection to project completion, as well as fostering reflective thinking. We strive to make ethics more than just a theoretical construct, but rather a tangible reality that resonates with diverse stakeholders. This accessible advice empowers future iGEMers to find their own real-world significance and sparks new perspectives for entrepreneurship, education and beyond.

To learn more about ethic, please visit HP.

Education

Public education and promotion in synthetic biology are indispensable components of our project. We rely not only on online promotion but more on face-to-face interactions with the general public. Our core mission not only focuses on the exploration of frontiers in synthetic biology but also the significance of public education and the promotion of comprehensive social progress, particularly in remote areas with relatively inadequate resources. During the summer vacation of 2024, we traveled to Wutai County, Shanxi Province, to conduct educational and promotional activities related to medical insurance and cancer prevention. Addtionally, we provided a week-long teaching support at the only primary school in the area. By bringing scientific knowledge to remote regions, we have expanded the scope and impact of Education and HP, making our project more inclusive and universally applicable. We believe that our efforts can serve as an inspiration for the broader iGEM community. Aware that the power of knowledge transcends geographical boundaries and brings hope and enlightenment to people in remote areas, we have carefully designed educational activities that are both scientifically rigorous and grounded in daily life. Our aim is to make complex medical knowledge accessible, comprehensible, and deeply ingrained through face-to-face interactions and communications.

While striving to make science accessible and fostering inclusivity by breaking down information barriers, we endeavor to achieve a harmonious fusion of science and art, enabling students and communities to engage with the world of synthetic biology through lens. Education is far more than a simplistic "I teach, you learn" process. We strive to provide personalized and meaningful learning experiences for all. We introduced mysterious bacterial friends to primary school students and received adorable "Play-Doh Bio-Targets". For middle school students, we opened the doors to laboratories and the wondrous world of synthetic biology for them, guiding them through the entire process of diagnosing and treating bacterial infections in the real world using a Problem-Based Learning (PBL) approach. For university students, we established the iBSRC Online Summer School, covering synthetic biology, academic guidance, and mentorship from renowned experts. We invited students from social science majors into the lab to evaluate projects and future challenges of synthetic biology from a uniquely non-STEM perspective. Leveraging China's National Cancer Prevention and Control Week, we conducted cancer awareness campaigns and free clinics in communities, correcting misconceptions and sparking further reflections on ethics and project development. The integration of multiple disciplines, cross-perspective interactions, multi-regional and multi-demographic outreach, and artistic expressions have contributed significantly to the success of our educational journey. Within the Education sector, we have provided detailed planning documents for most of our activities, making it easy for future iGEMers to replicate these initiatives. We wish to diversify the iGEM community, ensuring that more voices provide feedback on our strengths&weaknesses and illuminate the issues that synthetic biology must prioritize on its future path.

To learn more about our various education, please visit Education.