In the iGEM competition, safety has always been the most critical aspect of our work. As a high school team, we incorporated a dual suicide system into our project design: a cold-inducible system ensures that engineered bacteria function normally at the body temperature of 37 ℃, but automatically lyse when exposed to low temperatures outside the body; an arabinose-inducible system allows for artificial clearance of residual engineered bacteria inside the body through oral administration of L-arabinose, thereby minimizing the risk of environmental spread. Meanwhile, we strictly follow laboratory safety guidelines for water usage, electricity, and waste disposal, and actively promote the concept of “safety first” to the public, demonstrating our strong sense of responsibility to both science and society.

Ex Vivo – Cold-Inducible Suicide System

When the external environmental temperature falls below 16 ℃, the PcspA cold-shock protein promoter is activated and drives the expression of downstream T4 holin and T4 lysozyme. T4 Holin forms pores in the bacterial inner membrane, while T4 Lysozyme degrades the cell wall. Their synergistic action leads to rapid lysis and death of the engineered bacteria. As a result, while the bacteria can remain active and functional inside the human body (at ~37 ℃), they undergo self-destruction once released into external environments, thereby preventing unintended spread.

Figure 1: Design principle of the cold-inducible suicide system.

Experimental Validation

We first placed mRFP downstream of the PcspA promoter to evaluate its expression levels under different temperatures, followed by constructing a PcspA–T4 lysis engineered strain to monitor its growth under cold conditions. The results demonstrated that the PcspA promoter exhibited significant induction at low temperature: at 16 ℃, fluorescence intensity reached 270.84 ± 19.36 AU after 12 h, while at 37 ℃ it was only 67.92 ± 22.24 AU, showing a clear “on at low temperature, off at high temperature” pattern. Further testing with the PcspA–T4 lysis suicide system revealed that engineered bacteria displayed strong growth inhibition at 16 ℃, with OD600 dropping to 0.34 ± 0.08 at 8 h, compared to 0.83 ± 0.06 for the control strain. This indicates that cold exposure effectively triggers the lysis reaction. Collectively, these results confirm that PcspA is a reliable environmental sensing element and can successfully drive a suicide module to eliminate engineered bacteria at low temperatures.

Figure 2: Validation of the cold-inducible PcspA promoter and PcspA–T4 lysis suicide system (A: Temperature-dependent expression of mRFP driven by PcspA; B: Growth inhibition of engineered bacteria carrying PcspA–T4 lysis system at 16 ℃).

In Vivo – Arabinose-Inducible Suicide System

This system was designed based on the tightly regulated properties of the PBAD promoter. In the absence of arabinose, PBAD exhibits almost no basal expression. When exogenous L-arabinose is added, the AraC protein activates the promoter, initiating strong transcription of downstream genes. We placed T4 holin and T4 lysozyme (together forming the T4 lysis module) under the control of PBAD. Holin perforates the bacterial inner membrane, while Lysozyme degrades the cell wall. Their combined effect rapidly lyses the cells. Through this design, engineered bacteria can grow stably under normal culture conditions, but upon supplementation of L-arabinose, the suicide module is efficiently activated, enabling artificially controlled, rapid, and thorough clearance of bacteria in vivo. This strategy minimizes the potential risk of environmental release.

Figure 3: Design principle of the arabinose-inducible suicide system.

Experimental Validation

We first inserted mRFP downstream of the PBAD promoter to measure induction under different concentrations of L-arabinose. Next, the T4 lysis module was placed downstream of the same promoter to construct the arabinose-inducible suicide system. Results showed that PBAD had almost no background expression in the absence of arabinose, while its activity increased significantly with higher arabinose concentrations. At 0.5% arabinose, fluorescence intensity exceeded 1089 AU within 8 h. Based on this, PBAD–T4 lysis engineered bacteria were tested: after addition of arabinose at 4 h, rapid lysis occurred, and by 20 h, OD600 dropped to 0.19 ± 0.16, indicating almost no growth, whereas the control strain continued proliferating. In summary, the PBAD promoter exhibits strong dose-dependent induction and can effectively drive the suicide module to achieve artificially controlled lysis of engineered bacteria.

Figure 4: Validation of the arabinose-inducible PBAD promoter and PBAD–T4 lysis suicide system (A: Fluorescence report results of mRFP under PBAD; B: Lysis of engineered bacteria under PBAD–T4 lysis system upon arabinose induction).

During project implementation, our team encountered an issue regarding the source of phosphatidylcholine (PC) in culture medium. Since purified PC was not available for purchase, we planned to follow the literature and use egg yolk as a substitute source. To ensure that our experimental design complied with iGEM’s animal ethics and biosafety regulations, we proactively contacted the iGEM Safety and Security Committee before conducting the experiment. In our email, we explained in detail the purpose of the experiment, the methods to be used, and the source of materials—clarifying that only unfertilized, food-grade egg yolks would be used. We also requested the committee’s evaluation of the compliance of our experiment. This action reflects our team’s strong emphasis on research compliance and biosafety, as well as our responsible attitude of actively seeking official guidance whenever uncertainties arise.

Figure 5: Email communication with the iGEM Safety and Security Committee regarding the egg-yolk plate experiment.

Water Safety

Different types of water in the laboratory should be clearly distinguished in their usage: ultrapure water, distilled water, and tap water must never be mixed. Ultrapure and distilled water are reserved exclusively for experimental operations and instrument maintenance, and must not be consumed. When using a water bath, the water level should be kept appropriate to avoid dry heating or overflow. The power supply must be turned off promptly after use. In the event of water leakage, the water source should be immediately shut off and staff notified, in order to prevent equipment short-circuiting or floor flooding that could cause slipping accidents.

Figure 6: Caution – slippery floor hazard.

Electrical Safety

All electrical equipment in the laboratory must be operated only by trained personnel. Unauthorized rewiring or modification of electrical systems is strictly prohibited. High-power equipment (such as autoclaves and electrophoresis devices) should have independent grounding, and power cords and plugs must be checked for damage before use. Operators must ensure dry hands when handling sockets or switches and must never operate electrical devices with liquid containers nearby. After experiments, all power supplies should be switched off, and plugs should be removed if equipment will not be used for an extended period, in order to prevent accidents.

Figure 7: Laboratory electrical safety guidelines.

Waste Disposal

Waste generated during experiments must be collected and disposed of in a classified manner:

• Biological waste containing bacteria, plasmids, or genetically modified materials must be autoclaved before disposal.
• Liquid chemical waste should be collected in dedicated waste containers, categorized by type (acid, base, organic solvent, etc.), and handed over to certified disposal agencies.
• Sharps, such as broken glass and needles, must be discarded into designated sharps containers.

Direct disposal of laboratory waste into sinks or general trash bins is strictly forbidden, ensuring the safety of personnel and the protection of environmental biosafety.

Figure 8: Laboratory waste management procedures.

To promote the standardized development of synthetic biology, our team organized and hosted a roundtable discussion themed “Safe Experimental Design and Biosafety.” The event brought together representatives of teachers and students from our school to explore how the principle of “safety first” can be integrated into both research and practice. The discussion covered several key topics, including laboratory operation protocols, prevention of engineered bacterial leakage risks, and the design of suicide systems. Through open dialogue, participants not only shared experiences and challenges in experimental safety but also reached a consensus on the importance of incorporating safety considerations at the earliest stages of project design. This activity fostered interdisciplinary and cross-community dialogue, providing a practical case for students to understand and practice Responsible Research and Innovation (RRI) in synthetic biology. At the same time, it conveyed our team’s strong commitment to biosafety and social responsibility to the wider public.

Figure 9: Poster for biosafety promotion event.

Looking ahead, our team envisions further developing the engineered bacteria into a probiotic health product with potential application value. However, transforming laboratory results into products suitable for public use requires undergoing a series of rigorous safety and compliance evaluation procedures.

Toxicological and Biological Assessments

First, in vitro toxicology experiments and animal model studies must be conducted to verify the harmlessness of the engineered bacteria, as well as their stability and effectiveness inside the host. In addition, genetic stability testing and environmental risk assessments are essential to ensure that the engineered strain does not generate or disseminate antibiotic resistance genes, nor pose risks of environmental leakage.

Regulatory Compliance Pathway

Upon completion of scientific validation, the project must comply with the requirements of national food and drug supervisory authorities (such as China’s National Medical Products Administration (NMPA) or the U.S. Food and Drug Administration (FDA)). This process includes preclinical research, Phase I–III clinical trials, and final regulatory approval and market registration.

Manufacturing Standards

Furthermore, product manufacturing must follow Good Manufacturing Practice (GMP) standards to ensure traceability and quality consistency during large-scale production.

Only after completing these systematic safety assessments and compliance certification processes can engineered bacteria truly be introduced as a probiotic health product and contribute to human health.

Figure 10: Future safety work plan.