In the application of synthetic biology, safety is the cornerstone of project success. In our project, the modification of Escherichia coli to degrade glyphosate residues in tea plantations became the centerpiece of the study, and biosafety measures were key to ensuring that the process was harmless.

Glyphosate is a broad-spectrum herbicide that, with long-term use, can cause harm to both the environment and human health. First, glyphosate degrades soil, affecting the activity of beneficial microorganisms and disrupting soil structure, which in turn impacts crop growth. Second, excessive accumulation of glyphosate in the human body can lead to toxic reactions, impairing liver and kidney function, and it may pose risks to pregnant women and fetuses due to endocrine disruption. Additionally, glyphosate has been classified as a Group 2A carcinogen, indicating a potential cancer risk.

While the modified E. coli strain helps degrade glyphosate, its accidental release into the environment could pose various risks. First, the strain could spread in the environment, affecting ecosystems. Second, the strain’s genes could be transferred horizontally to other microorganisms, increasing genetic mobility and posing potential ecological and biosafety hazards. Therefore, preventing the spread of the modified strain and gene leakage into the environment is a core consideration in our project design.

In this project, we designed a temperature-induced suicide system to ensure that the modified E. coli strain cannot survive for long outside the laboratory, preventing gene leakage. Specifically, we utilized the cold-induced promoter pCspA , which activates when the environmental temperature drops below 37°C. The promoter controls the expression of the suicide gene mazF . When E. coli enters a natural environment where the temperature is below 37°C, the cold-induced promoter activates, triggering the expression of mazF and leading to the engineered bacteria’s cell death. This design effectively prevents the spread of the modified strain in open environments.

Additionally, the activation of this suicide system has a time delay window. During this window, the engineered bacteria can complete their task of degrading glyphosate before automatically dying once the suicide system activates, ensuring that the bacteria do not persist in the environment after completing their task. In this way, our design not only ensures effective glyphosate degradation but also further guarantees biosafety by preventing gene pollution in the environment.

In laboratory management, we established strict safety protocols and operating guidelines. All laboratory personnel must undergo comprehensive safety training before starting work to familiarize themselves with emergency response measures. Specifically, protective clothing, gloves, and safety goggles must be worn before experiments.

All personnel involved in experiments must undergo comprehensive safety training before starting. This training covers basic safety requirements for experimental operations, emergency response measures in case of accidents, and proper handling of experimental waste. Through this training, we ensure that every member is familiar with laboratory safety regulations and can respond appropriately in emergencies.

We developed safety measures for handling experimental waste, particularly modified strains, to ensure that they do not harm the environment. In the event of a spill involving contaminated reagents, designated cleaning agents and protective equipment must be used immediately, and the incident must be reported to the laboratory supervisor. Specific waste disposal processes are established for different types of experiments to ensure that all experimental waste is handled safely and harmlessly.

To ensure biosafety, strict access control is implemented in the laboratory. Only authorized personnel are allowed to enter the laboratory area, and unauthorized personnel are prohibited from entering without permission. This measure effectively prevents untrained or unfamiliar team members from entering the laboratory, reducing safety risks.

We maintain detailed records of all experimental operations to ensure traceability. Each experiment’s steps, strains, materials used, and results are fully documented to prevent misuse or data loss. This rigorous record-keeping system helps to identify and correct problems in a timely manner and ensures the safety of the experiments.

To ensure the biosafety of the project, we adopted multi-layered safety measures. In the laboratory, strict personal protection requirements, comprehensive safety training, and detailed record-keeping ensure that all personnel operate safely, preventing accidents. The implementation of laboratory access control measures further restricts unauthorized personnel from entering the lab, reducing potential risks.

Additionally, we designed a temperature-induced suicide system to address the environmental risks associated with the modified E. coli strain. This system ensures that the strain automatically dies after completing its task, preventing its spread in open environments and gene leakage.

The combination of these safety designs and management measures ensures that both the experimental process and environmental impact remain under control, providing strong support for the project’s successful execution.