Wet lab

We have successfully developed a novel synthetic biology-based "smart" bacterial therapeutic platform designed to address core challenges in colorectal cancer immunotherapy. This platform employs engineered E. coli as a delivery vehicle, which specifically senses the hypoxic tumor microenvironment to precisely activate the host STING immune pathway. Furthermore, it utilizes c-di-GMP-induced biofilm formation to achieve long-term bacterial colonization at the tumor site. Our work presents a highly promising, efficient, low-toxicity, and orally administrable minimally invasive strategy to overcome key obstacles, including low drug delivery efficiency, the resilient immunosuppressive microenvironment, and the urgent need to improve patient quality of life.

Current Landscape and Pressing Challenges in Colorectal Cancer Therapy

Despite continuous advances in colorectal cancer treatment, clinical practice, particularly for patients with advanced and metastatic disease, still faces severe challenges:

1. Low Response Rate to Immunotherapy: Over 95% of colorectal cancers are "cold tumors," characterized by a highly immunosuppressive microenvironment that renders monotherapies like PD-1/PD-L1 checkpoint inhibitors largely ineffective.
2. Challenges in Drug Delivery and Retention: The physical barriers and abnormal vasculature of solid tumors prevent systemically administered chemotherapeutic and targeted drugs from effectively penetrating and accumulating in the tumor core. Small molecule drugs, such as exogenous STING agonists, also suffer from short half-lives and rapid clearance.
3. Treatment-Related Toxicity and Off-Target Effects: The systemic toxicity of chemotherapy and radiotherapy significantly impairs patients' quality of life. Even targeted drugs can cause off-target effects on healthy tissues when administered systemically.
4. The Pressing Need for Organ Preservation: For patients with low rectal cancer, conventional radiotherapy and surgery often result in permanent stomas, severely compromising bowel function and personal dignity. Consequently, minimally invasive therapies that can preserve organ function represent a critical unmet clinical need.

Our Innovative Solution: An Intelligently Responsive Engineered Bacterial Platform

To address the above challenges, we designed and constructed a multifunctional, integrated engineered E. coli strain. Its innovativeness is manifested at three key levels:

1. Precision Core Logic: Hypoxia Sensing and Targeted Activation: We exploit the characteristic hypoxic microenvironment within tumors as a natural "switch" to tightly control the expression of the c-di-GMP synthase. This design ensures that the therapeutic command is initiated specifically at the tumor site, minimizing damage to healthy tissues and achieving unprecedented precision and safety. After the engineered bacteria are phagocytosed by tumor-associated macrophages, the intracellularly produced c-di-GMP efficiently and directly activates the STING pathway from within the cell. This "Trojan Horse" activation strategy proves more effective than exogenous drug administration.

   We exploit the characteristic hypoxic microenvironment within tumors as a natural "switch" to tightly control the expression of the c-di-GMP synthase. This design ensures that the therapeutic command is initiated specifically at the tumor site, minimizing damage to healthy tissues and achieving unprecedented precision and safety. After the engineered bacteria are phagocytosed by tumor-associated macrophages, the intracellularly produced c-di-GMP efficiently and directly activates the STING pathway from within the cell. This "Trojan Horse" activation strategy proves more effective than exogenous drug administration.

2. Unique Colonization Advantage: Biofilm-Mediated Long-Term Retention: Our design ingeniously capitalizes on the dual function of c-di-GMP: it acts not only as an immune messenger but also as an inducer of biofilm formation. High levels of c-di-GMP expression drive the engineered bacteria to form biofilms on and within the tumor. This allows the bacteria, much like a "Trojan Horse," to effectively resist clearance by intestinal peristalsis and achieve long-term colonization, enabling continuous release of immune-stimulatory signals and overcoming the challenge of short retention time for conventional drugs in the dynamic intestinal environment.

3. Critical Safety and Efficacy Control: An Engineered Multi-Layered System: To ensure robust biosafety while maintaining therapeutic efficacy, we implemented a dual-layer containment strategy combining metabolic constraints and inducible control.

   First, we established a thymidine-dependent auxotrophic strain through thyA gene knockout, encoding thymidylate synthase. This design creates a fundamental biological containment: the engineered bacteria can only replicate in thymidine-rich environments like tumors, where nucleotides are abundantly available, while any escaped bacteria cannot proliferate in normal tissues or the external environment. This intrinsic metabolic limitation provides continuous, environment-responsive safety without external intervention.

   Second, we integrated an inducible suicide system that serves dual purposes. For safety, it acts as a programmable "kill switch" that can be chemically activated to eliminate bacteria when needed. For efficacy, it enables controlled bacterial lysis to enhance c-di-GMP release efficiency at the tumor site, ensuring optimal immune activation.

Together, these systems create a sophisticated safety architecture where the thymidine auxotrophy provides passive, continuous containment and the inducible system offers active, timed control - representing an optimal balance between safety assurance and therapeutic performance.

Project Contribution and Future Perspectives

The contribution of this iGEM project extends far beyond the construction of a single genetic circuit; it represents a critical step forward in the development of next-generation live biotherapeutic products:

1. Scientific Paradigm Innovation: We have established a novel scientific paradigm by modularly integrating four core functions into a unified engineered E. coli system. This integration begins with the hypoxia-responsive c-di-GMP expression plasmid pUCP20-GmR-P_fnrS-yedQ(BBa_25WD29E5), where the oxygen-sensitive promoter P_fnrS(BBa_25DMNRIB) drives the expression of the diguanylate cyclase YedQ, enabling tumor-specific production of the immunostimulatory molecule c-di-GMP. This is synergistically combined with a programmable lytic safety circuit (pTBR2iB-BBa_2555BR7G), which incorporates a quorum-sensing LuxR-P_luxI module to control the expression of the lytic effector PhlA, allowing for inducible bacterial clearance. The system is further secured by a thymidine auxotrophic chassis (thyA knockout), ensuring environmental biocontainment. This multi-layered design—coupling environmental sensing (hypoxia), immune activation (STING), tumor retention (biofilm), and programmable safety (lysis + auxotrophy)—provides a comprehensive new blueprint for live bacterial therapies and deepens our understanding of local immunomodulation in the gut.
2. Technical Pathway Breakthrough: Our project demonstrates a significant technical breakthrough by maximizing functional output through refined genetic tool selection and rational circuit design. We achieved high-level, hypoxia-triggered c-di-GMP production by employing the high-copy-number pUCP20-GmR backbone for the P_fnrS-yedQ expression cassette, ensuring strong gene dosage and robust STING activation upon bacterial uptake by macrophages. Furthermore, we validated the dual functionality of c-di-GMP—acting both as an intracellular STING agonist and an extracellular matrix inducer for biofilm-mediated tumor retention—using a single, optimized genetic module. For controlled biocontainment, we implemented an AHL-inducible kill switch (pTBR2iB), which leverages a positive feedback loop (P_luxR-driven luxR expression) to amplify sensitivity and ensure rapid, synchronized lysis via PhlA. This precise engineering, from promoter strength to circuit dynamics, showcases an efficient and sophisticated design paradigm for synthetic biology, turning a single bacterial signal (c-di-GMP) into a multi-functional therapeutic output.
3. Rigorous Engineering Cycle Implementation: Our project was developed through a rigorous engineering cycle of Design-Build-Test-Learn, with biosafety as a central consideration from the outset. In the design phase, we selected the thyA knockout as our primary biocontainment strategy after evaluating multiple essential gene targets. This decision was based on its proven efficacy in clinical-stage strains like VNP20009, single-pathway essentiality. During the build phase, we constructed the auxotrophic chassis and subsequently introduced our therapeutic and safety modules. This iterative process led us to a final, optimized design where metabolic constraints, therapeutic efficacy, and multi-layered biocontainment (combining auxotrophy with an inducible suicide system) are cohesively integrated, embodying the highest standards of responsible and effective engineering.
4. Clinical Translation Value: Our platform heralds a potentially transformative treatment modality. Via oral or local administration, it offers a promising, efficient, low-toxicity, minimally invasive therapeutic option that could significantly improve the quality of life and aid in organ preservation for colorectal cancer patients who are ineligible for surgery or unresponsive to existing immunotherapies.
5. Parts Contribution: Our team has made substantial contributions to the iGEM Parts Registry. We newly uploaded 12 Basic Parts, enriching the available genetic components for synthetic biology research. Leveraging these newly uploaded Basic Parts along with existing ones, we successfully constructed 4 Composite Parts. As seen in the wiki page https://2025.igem.wiki/sustech-med/parts, these Composite Parts include plasmids like pTBR2iB(BBa_2555BR7G), which implements an AHL-inducible kill switch in E. coli for controlled cell lysis; pUCP20-GmR-P_fnrS-yedQ(BBa_25WD29E5), a hypoxia-responsive c-di-GMP expression plasmid enabling tumor-specific production of c-di-GMP; and pGL3-P_fnrS, a hypoxia-responsive luciferase expression plasmid to verify promoter function. These Composite Parts serve as valuable tools, each with specific functionalities in areas such as bacterial safety control, secondary messenger production under hypoxic conditions, and promoter function verification, thereby advancing the field's capacity for engineering complex biological systems.

In conclusion, our research is not merely a successful synthetic biology exercise but a targeted response to a pressing clinical need. We firmly believe that this work lays a solid foundation for translating 'smart bacteria' from a laboratory concept into future clinical reality, bringing new hope and direction for ultimately overcoming colorectal cancer.

Models

Inducer Diffusion Quantitative Model

Our model provides the iGEM community with a validated, modular toolkit and design philosophy for quantitative modeling, aimed at helping future teams more efficiently analyze, predict, and optimize their engineered biological systems.

First, we contribute a general quantitative analysis framework based on the Hill function for reaction optimization. We demonstrate how to model the common dose-response relationships in biological systems, and future iGEM teams can easily apply this framework by identifying key regulatory steps, collecting a minimal set of wet lab data at a few concentrations, and using our provided parameter-fitting methods to calibrate the model. This process accurately determines crucial parameters like the half-activation constant and the Hill coefficient. Once calibrated, the model can predict the system's response at any reagent concentration, allowing teams to computationally determine the optimal conditions for achieving their goals, such as maximizing product yield or minimizing response time. This significantly reduces the need for tedious trial-and-error experiments, saving time and resources and enabling a more efficient, model-driven approach to synthetic biology.

Secondly, our project establishes a model for molecular diffusion in 3D space and provides a "methodology library" with two distinct methods for solving its governing partial differential equations (PDEs): the classic Finite Difference Method (FDM) and the cutting-edge Physics-Informed Neural Networks (PINNs). This dual-method approach serves as a design philosophy for building sophisticated spatiotemporal models, allowing teams working on projects with spatial dimensions, such as cell-to-cell communication or drug release, to select the tool best suited to their needs. For systems with regular geometries, our intuitive FDM implementation provides an accessible starting point, while for projects involving complex boundaries or sparse data, our powerful PINNs framework offers a more advanced solution. By providing this scalable modeling pathway from simple to complex, we empower future teams to build dynamic models that more closely approximate real biological scenarios, possess stronger predictive power, and ultimately enhance the depth and impact of their projects.

Individual-based Microbial Community Development Model:

1. Design optimization: We built a publicly available CompuCell3D model simulating oxygen-dependent bacterial adhesion. Future teams can reuse it to tune promoter sensitivity or sensing strength based on model feedback, iteratively updating parameters with wet-lab data.

   This helps achieve optimal adhesion strength and higher tumor-specific binding, reducing bacterial dosage needs.

2. Extendable “toy framework”: The model can be easily adapted to other environmental cues (e.g., pH, nutrients, or signaling) or adhesion mechanisms (e.g., receptor–ligand systems), providing a simple framework for testing environment-dependent cellular behaviors.

3. Modeling translation method: We also provide a conceptual example of translating biology into computational rules—extracting key processes like sensing, signaling, motility, and secretion into code—offering a reusable strategy for future iGEM modeling teams.

Hardware

Our team has designed a three-layer microfluidic colorectal organoid-on-a-chip, providing future iGEM teams with a platform that closely simulates the human colon environment. Our work is innovative both theoretically and practically. Here are our specific contributions:

Firstly, We have added comprehensive documentation to the existing microfluidic chip section on the registry page. This includes detailed descriptions of the chip's design principles, fabrication processes, and operational procedures, from the preparation of 3D-printed molds to the assembly of PDMS-based microfluidic chips. Additionally, we have documented methods for the 3D construction and culture of patient-derived colorectal organoids, as well as their 2D conversion, differentiation induction, and seeding onto the chip.

While we have not directly improved existing software or hardware tools, our design and documentation lay the groundwork for potential future enhancements. For instance, our chip design could facilitate the development of new hardware tools to simulate and analyze biological processes within the chip.

We have documented potential issues encountered during chip fabrication and organoid culture, along with their solutions. This information will be invaluable to future teams facing similar challenges.

Secondly, Our PDMS molds for the microfluidic chip were prepared using 3D printing and other techniques, with iterative samples and SolidWorks drawings provided. We have thoroughly documented the chip fabrication process, including mold design, material selection, and post-processing steps, to assist future teams in replicating our work or making further improvements.

We believe our work will be a valuable resource for future iGEM teams, aiding in a better understanding and application of microfluidic technology and organoid models. We encourage future teams to innovate upon our work to advance the field.

Inclusivity

This year, we focused on the elderly community, aiming to gain a deeper understanding of their challenges in accessing science, their needs related to it, and to help them learn more about it.

Here we conclude what we have learned. The majority of elderly people in China don’t like to actively participate in scientific research. Nor do most of them really want to learn “science” in the way we usually imagine it. However, there are specific areas of science that they are genuinely interested in. They want to learn about health protection, anti-fraud, and parenting.

Thus, our inclusivity work focused on their needs, making sure what we did was what they wanted, instead of what we wanted them to know. We believe this principle works for all iGEMers concerning public engagement. We strongly encourage our fellow iGEMers to respect the needs of their target groups, and base their efforts on concrete investigations. As for the elderly community, we suggest future teams further explore their barriers to science and do more to help them learn. We emphasize that this is not only about scientific literacy itself, but also about the well-being of elderly people, especially given the overwhelming amount of false information and scams targeting them today.

During our work with the elderly community, there was a very specific group that caught our attention — the illiterate population. We first noticed them in our community activities. It was already a shock to discover that some people in our country still cannot read, but only after talking to them did we realize how illiteracy remains an unsolved problem in China. According to our interviews, illiteracy strongly influences their quality of life. Daily tasks like using smartphones, communicating with people from other regions, or simply going out can be extremely difficult. What’s even more surprising is that they all know many others who are also illiterate, suggesting that this is not a rare situation. When asked if they were willing to learn how to read, they often expressed the intention to learn but gave a common response of “no”, saying they were too old or that learning to read was simply too much for them.

We believe illiteracy is an extremely overlooked problem, at least in China. We encourage future teams to pay attention to this issue, to deeply investigate this group of people, to truly understand their situation, their barriers, possible ways to help them, and most importantly, their needs. We hope this group can be seen by more people and receive the help they need, not only to learn more about science, but also to live a better life.

Integrated human practice

We believe that the future of synthetic biology depends not only on scientific progress but also on trust and understanding between doctors and patients. To address this, we created a white paper on physician–patient communication, offering practical guidance for healthcare professionals and patients while also serving as a resource for future iGEM teams engaging with medical stakeholders. Our mission is to strengthen communication, build trust, and provide the iGEM community with tools to connect more responsibly and meaningfully with the people their innovations are designed to help.