This project aims to provide directly referable contributions for future iGEM teams, with a core focus on using and improving the existing Bronze Part—MazF (BBa_K1096002). We experimentally validated its application in a biocontainment system and delivered comprehensive documentation updates. We constructed a colorectal cancer therapeutic system based on engineered probiotics, integrating a neohesperidin biosynthesis pathway and a dual suicide safety mechanism. Within this system, MazF serves as the key toxic component, placed under the control of both arabinose-inducible (pBAD) and cold-inducible (pCspA) promoters to achieve controllable cell clearance. Through this work, we not only confirmed the high efficiency and reliability of MazF, but also provided a standardized case for its safe application in synthetic biology, enabling future teams to rapidly design biocontainment strategies. Additionally, we developed a colorectal cancer prevention handbook to extend our societal impact.
We have made significant contributions to the Bronze Part MazF (BBa_K1096002), originally submitted by the iGEM13_ITU_MOBGAM_Turkey team. MazF is an RNA endonuclease toxin derived from Escherichia coli that specifically cleaves mRNA at ACA sequences, leading to the interruption of protein synthesis and ultimately cell death. In this project, we integrated MazF into the biosafety module of a colorectal cancer therapeutic system for the first time, and conducted comprehensive functional validation and documentation updates.
Application of MazF in the Biocontainment System
We placed MazF under the control of two environmentally inducible promoters:
Arabinose-inducible system (pBAD-MazF): Allows patients to trigger bacterial self-destruction in vivo via oral intake of arabinose.
Cold-inducible system (pCspA-MazF): Ensures engineered bacteria automatically lyse under low-temperature conditions in vitro, preventing genetic escape.
Figure 1. Genetic circuit diagram of the MazF-based suicide system.
By expressing MazF in E. coli BL21 and EcN strains, we confirmed that:
Under induction conditions (e.g., 0.2% arabinose or 16°C), MazF expression led to a significant decrease in OD600 (over 80% lysis rate), with complete inhibition of cell growth. The control groups (without induction) showed minimal background expression, demonstrating the tight controllability of the system.
Figure 2. Growth of engineered bacterial strains with the in vivo suicide system under different arabinose concentrations. Caption: Comparison of growth curves of the in vivo suicide system strain under 0% and 0.2% arabinose conditions.
Figure 3. Growth of engineered bacterial strains with the in vitro suicide system under low temperature. Caption: Comparison of growth curves of the in vitro suicide system strain and wild-type strain at 16°C.
We added the following content to the MazF page on the Part Registry:
- Detailed experimental data (including growth curves and lysis efficiency plots).
- Application examples (e.g., circuit diagrams of dual suicide systems).
- Potential future directions (e.g., combining with other sensors for multi-condition triggering).
Key Achievements and Future Value
- Standardized Safety Module: The validation of MazF provides future teams with a "plug-and-play" biosafety tool that can be rapidly integrated into projects such as probiotic therapies and environmental remediation.
- Risk Reduction: The dual induction mechanism significantly reduces the risk of engineered bacteria escaping, aligning with biosafety regulatory requirements.
- Enhanced Documentation: The updated documentation includes sequence optimization suggestions, application scenarios, and troubleshooting guidelines, helping teams avoid redundant work.
We created a Colorectal Cancer Prevention Handbook to provide preventive guidance for individuals at potential risk of colorectal cancer, demonstrating how iGEM projects can bridge scientific innovation and societal needs.
Prevention of Colorectal Cancer.pdf
Our core contribution lies in the in-depth utilization and documentation enhancement of the Bronze Part MazF, directly helping future iGEM teams save time and reduce risks in biosafety design. By integrating MazF into an innovative therapeutic system, we validated its reliability as a "safety switch" and contributed new experimental data and application cases to the Part Registry. Together, this work establishes an expandable reference framework, advancing synthetic biology toward safer and more efficient development.
