Biosafety is the top priority of our iGEM project, as failing to comply with relevant measures can lead to serious consequences such as disease outbreaks, environmental contamination, or decreased experimental reliability. To ensure biosafety in the build-up of our design, we designed a suicide system, promoted biosafety ideas in the people around us, and adhered to rigorous biosafety protocols under Biosafety Level 1 (BSL-1).

Even as students, we recognize that a strong commitment to biosafety is fundamental to responsible scientific innovation, since fostering robust biosafety awareness is essential not only for accident prevention in the lab but also for research integrity and efficiency overall. This is why our team took the following actions to strengthen biosafety in our project.

Strain Selection

We considered safety in every step of our project design. For our project, we chose to use E. coli Nissle 1917 (EcN) as our chassis organism, a member of the white list, which also classified as biosafety level 1(BSL-1). This specific strain of E. coli bacteria carries many advantages that are crucial for the development of our project. Specifically, EcN is non-toxic, non-pathogenic, and has a long clinical history as a therapeutic agent for gastrointestinal diseases. It can colonize the human gut in a stable yet controlled matter, which is crucial in remediating colorectal cancer. Finally, the genome of this strain has been fully sequenced, allowing genetic modifications with minimized risks. All of these features make E. coli Nissle 1917 a reliable and effective delivery vehicle for our project.

Suicide System

It is important to prevent our engineered bacteria from harming its host cell or damaging its environment. This is why our team, just like many iGEM teams in the past, developed a kill-switch circuit to ensure the safety of our contraption.

Before use, our engineered bacteria are in a controlled environment with arabinose and a 660-nm red light. These two parts of our project are crucial in the construction of our suicide system.

Figure 1 Suicide System

During the therapeutic stage, the two signals (arabinose and 660-nm red light) activate regulatory that allow the expression of MazE, an antitoxin that neutralizes the toxin. In turn, the bacteria is able to survive and achieve therapeutic functions.

At the end of the therapeutic stage, the suppository detaches into two: a head with the red light and the tail with the bacteria. At the same time, there is no arabinose left. Without these two signals, MazE is no longer produced, leaving toxin MazF active. This triggers the self-killing of the bacteria, preventing uncontrolled survival after therapy.

Comparative Analysis and Self-Optimization of Suicide Systems

To improve our design, we looked through many team wikis from iGEM teams in the past. Specifically, we mainly analyzed systems that had similar features as our team design.

Fudan University's (Team: Fudan/Design - 2020.igem.org, n.d.) Kill Switch consists of a toxin/antitoxin system MazF/MazE and an RNA thermometer NoChill-06 to regulate it to deprive of the survivability of engineered Nissle in the environment when excreted from the human intestine. The antitoxin MazE is liable and expressed at a relatively high level. The MazF toxin is constitutively co-expressed with the antitoxin under the control of an RNA thermometer NoChill-06. When the temperature is equal to or higher than 37°C, the constitution promoter will trigger the system, NoChill-06 unfolds and exposes its ribosome binding site (RBS) to express. MazE and MazF neutralize each other by protein-protein interaction and form a stable complexity in a one-to-two ratio; when the bacteria encounter a cold shock (30℃), MazE is degraded rapidly by an ATP-dependent serine protease ClpAP and releases MazF. The toxin MazF acts as a site-specific endoribonuclease to almost all cellular mRNAs, resulting in cell growth arrest and finally cell death. Their design carries several advantages: Despite the restricted living condition of the engineered bacteria, their overall design is effective in controlling the survival of engineered bacteria that exit the designed environment following the designed procedure. However, there are certain features of their projects that may be considered defects: They pointed out that “once the Kill Switch was implanted, the engineered bacteria could no longer be frozen as a glycerol stock, and it must be maintained in the culture media above 30 degrees.” The small defined range of temperatures in which the engineered bacteria can live might not be advantageous to this project.

Figure 2 Team Fudan's Suicide System Design

The team Pasteur Paris (Team: Pasteur Paris - 2018.igem.org, n.d.) uses a kill switch based on temperature: it enables bacteria to survive at human body temperature (37°C) but die at lower temperatures (under 22°C). They use toxin/antitoxin couple CcdB/CcdA. The toxin targets and inhibits the GyrA subunit of DNA gyrase, an essential bacterial enzyme that catalyzes the super-coiling of double-stranded closed circular DNA. At 37°C, the quantity of antitoxin CcdA is high enough to cope with the leaky low level of toxin produced. However, if the bacteria happen to be in an environment at a lower temperature, the toxin promoter is not repressed anymore, the quantity of toxin becomes too important, and the bacteria is not able to grow. In their designed environment, we can infer based on their design that when the temperature is between 37°C and 22°C, instead of dying immediately, the bacteria will gradually die or will not be in a very active state, making them somewhat resistant to accidents such as when the person drinks water to take drugs, the temperature will be lower than 37°C for a short time. However, their bacteria, just like that of Fudan's team, also only works in specific temperature ranges.

Figure 3 Function of the kill switch under 37°C and 22°C

The NEU China A team (Team: NEU China A - 2018.igem.org, n.d.) designed a cold shock kill switch based on the toxin-antitoxin system-mazEF- a natural toxin-antitoxin system found in E. coli. MazF is a stable toxin protein, and mazE is an unstable antitoxin protein. The team used a mRNA that is mediated by the cold-acting promoter CspA and can only be efficiently translated at a low temperature of, for example, 16 °C to activate the expression of mazF at low temperatures. As seen in the graph.

Figure 4 A cold shock kill switch based on the toxin-antitoxin system-mazEF- a natural toxin-antitoxin system found in E. coli.

In 2019, team NEU CHINA used the same kill-switch design as team NEU China A used in 2018 but made some improvements by palliating the symptom of severe MazF leakage problem. They introduced MazE to reduce the toxicity of MazF and experimented to test it.

Figure 5 Schematic Design of the Kill Switch. PcspA, a cold-acting promoter which can only be activated at16'C.PT7, the gene downstream of this promoter will be transcribed when there is T7 RNA polymerase. MazF, a stabletoxic protein, under the control of the T7 promoter, which can kill the bacteria at low temperature. MazE, an unstableantitoxic protein, which prevents MazF from killing the bacteria at 37 °C.

Their system is well-organized, and the bacteria will be unlikely to survive once it leaves the designed environment under the ideal circumstances.

However, if the patient chooses to stop his/her treatment, there might be some engineered bacteria left behind.

Comprehensive Advantages of the System and Future Prospects

Our system, by integrating the strengths of various teams, has developed a unique safety advantage: it uses red light signals with better tissue penetration, enhancing the feasibility of in vivo applications; dual-signal cooperative control provides higher reliability; and the EcN chassis ensures good biocompatibility. Although our system is more complex than some basic designs, through modular design and experimental verification, stable operation has been achieved.

In the future, we will continue to optimize the signal response efficiency and dynamic range, and at the same time consider introducing more environmental response components to enable the system to better adapt to the complex in vivo environment and lay a more solid foundation for clinical transformation. We will also continue to draw on the design concepts of outstanding teams to continuously improve the safety performance of the project.

Our team developed a comprehensive set of designed to rigorously assess essential knowledge and ensure clear understanding of critical safety practices. In parallel, we created and disseminated visually engaging laboratory safety posters to raise public awareness and reinforce key messages. Building on these efforts, we organized a series of practical safety education initiatives, including biosafety gatherings, the distribution of detailed safety manuals, and the planning of public lectures to broaden understanding of biosafety principles. Importantly, recognizing that China’s current legal framework for biosafety remains underdeveloped, we also submitted a policy proposal to government authorities outlining recommended improvements to the Biosafety Law.

Figure 6 Poster for the laboratory safety lecture

We ensured safety within our lab by going through extensive training beforehand and by implementing strict protocols during experiments.

During our time in the lab, we had several research supervisors watch us conduct all of our experiments. By doing so, we decreased our risk of operational errors substantially.

We implemented strict sanitation and clean-up measures within the lab by separating hazardous materials from domestic waste into different trash bins. This not only helped our lab recycle experimental items but also enhanced the overall safety of our procedures.

Experiments that involve the use of hazardous materials happen in isolated areas. For example, all cell related experiments are conducted in a clean bench away from other experiment areas, while experiments such as the gel electrophoresis are conducted under a fume hood. This prevents contamination and is closely aligned with the safety protocols of our lab.

All personnel that enter our lab are required to wear non-sterile gloves, lab-coats, long pants, and tennis shoes. We learned how to handle unlikely emergency events, what different caution signs in the lab meant, and aseptic techniques to use during our wet lab process.

Figure 7 Laboratory safety sign

During our project design stage, we originally planned to use a hemolysin-related protein in our system. However, considering its potential biosafety risks, we proactively contacted the iGEM Safety Committee for guidance. After reviewing our proposal, the committee informed us that hemolysin is not included in the iGEM whitelist, and therefore cannot be used by high school teams. Following their advice, we immediately decided to remove this component from our design to ensure full compliance with iGEM’s safety policies and uphold the highest standards of responsible research.

Figure 7. Official email reply from the iGEM Safety Committee confirming that hemolysin is not included in the whitelist for high school teams.

Our project prioritized biosafety from the start by using E. coli Nissle 1917 (EcN), a non-pathogenic BSL-1 strain with a strong safety record. On this chassis we built a dual-signal suicide system regulated by arabinose and 660-nm red light, ensuring bacteria survive only under controlled therapeutic conditions and self-eliminate afterward. Compared with earlier systems relying on single signals or environmental cues, our design provides stronger stability and in-vivo applicability.

In parallel, we developed a rigorous laboratory safety framework. This included clear safety management procedures, recognition and training on laboratory safety signs, and Q&A exercises to test essential knowledge. To extend impact beyond the lab, we created posters, organized biosafety workshops and public lectures, and distributed manuals. We also submitted policy suggestions to improve China’s Biosafety Law, highlighting our commitment to safety at both the experimental and societal levels.