Safety

Instruction

Safety has always been our top priority. Prior to conducting experiments, all team members received rigorous laboratory safety training, and safe practices were maintained throughout the entire experimental process. In our project, engineered bacteria will be applied in wastewater treatment. However, their potential dispersal with water flow poses a risk of leakage. To address this, we implemented effective safety measures, such as incorporating kill switches and enhancing physical interception in both the engineered bacteria and laboratory safety facilities.

1. Physical Interception

In this project, our biosafety control strategy primarily relies on physical interception methods. The environment of a sewage treatment plant functions much like a closed container. Combined with the filtration and supporting facilities positioned before and after the treatment units, this creates a relatively sealed system. As a result, the likelihood of engineered microorganisms “escaping” into the surrounding environment is extremely low.

The physical interception works by keeping the immobilized microorganisms trapped inside the carrier. Because the biological aerated filter functions like a relatively closed system, it lowers the risk of microorganisms being washed out with the treated water. At the same time, it improves contact and exchange between microorganisms and their substrates, which boosts treatment efficiency while reducing ecological risks.

Physical interception serves as a fundamental safeguard within the biosafety module, designed to limit the spatial movement of engineered microorganisms. By confining them within physical barriers, it prevents their escape from controlled treatment systems (such as wastewater facilities) into natural ecosystems, thereby mitigating potential ecological risks.

For instance, the Beijing Engineering Research Center for Advanced Wastewater Treatment Technology, affiliated with Peking University, has demonstrated relevant applications in biochemical wastewater treatment. This center developed a polyurethane-based macroporous reticular functional carrier for immobilized microorganisms, integrated with an aeration biological filter.

By creating a polyurethane carrier with a porous, interconnected structure (pore size 0.5–1.5 mm), the system works seamlessly with the aeration filter. This setup allows nitrification and denitrification to occur at the same time, strengthens microorganisms’ resistance to free ammonia, and removes the need for backwashing.

Fig.1 | The immobilization of engineered bacteria is achieved within a closed system
by employing a polyurethane-based macroporous reticular functional carrier, which prevents bacterial leakage

2. Suicide switch

Additionally, our team incorporated a temperature-sensitive promoter in our biosafety module to prevent accidental survival and leakage of engineered bacteria in the environment. The design, first created by the UCAS iGEM team in 2020, is based on a cold-inducible switch constructed from the temperature-sensitive transcriptional repressor CI434 and TEV protease. We replaced the SsrA tag into the LAA-LAA tag, aiming to improve the efficiency of the suicide switch.

At temperatures above 37°C (during wastewater pretreatment by heating), CI434 is constitutively expressed and represses the pCI434 promoter, thereby blocking TEV protease production. Although the downstream Doc toxin is transcribed, its LAA-LAA tag degradation tag ensures that the toxin is quickly broken down, preventing accumulation and maintaining a safe, non-toxic state.

However, when the temperature drops to around 34-37°C (bacterial leakage into natural environments or the human body surface), TEV protease is expressed and becomes active. TEV protease cleaves CI434, inactivating it and lifting the repression of the pCI434 promoter. This establishes a positive feedback loop where TEV expression is further enhanced with CI434 inactivation. At the same time, TEV protease cleaves the engineered SsrA hybrid tag fused to Doc toxin, removing the LAA-LAA tag. The stabilized Doc toxin accumulates within the cell, triggering its toxic effect and leading to cell death.

This temperature-dependent kill switch ensures that engineered microbes remain harmless during controlled, high-temperature wastewater treatment but are efficiently eliminated if they escape into cooler external environments, building a robust guard against unintended gene leakage.

Fig.2 | Design of the Genetic Circuit for the Suicide System

3. Lab safety

In addition to constructing genetic protection, we focus on extensive laboratory safety protocols. All members are trained in proper handling techniques, such as how to handle culture transfer without cross-contamination and maintaining clean and tidy work areas. We label all samples and equipment clearly to prevent mistakes and maintain traceability in our workflow. Most of the work is conducted on special compartments, and when needed, inside biosafety cabinets that help retain the microorganisms. These procedures ensure that engineered bacteria do not escape controlled environments.

Fig. 3 | Our biosafety cabinet, where most sensitive experimental operations are performed under sterile conditions.

Members also clean their workspaces with ethanol before and after experiments and sterilize apparatus such as pipettes and containers to prevent cross-contamination. Gloves and lab coats are worn at all times, and hands are washed thoroughly once a member is out of the lab. We implement strict lab entry and exit procedures, involving cleaning areas, throwing away garbage properly, and keeping all cultures locked away so nothing accidentally escapes from the lab. Following these practices regularly will protect both the researchers and the environment while ensuring accurate experimental results.

Fig. 4 | Our sterilizer, used to sterilize equipment and materials, ensuring a contamination-free environment.