To ensure the safety and integrity of our experimental process, we have implemented comprehensive risk management measures and established strict guidelines for team members. Basic safety protocols, such as wearing personal protective equipment (PPE) and prohibiting food and drinks in the laboratory, are strictly enforced. Proper handling of experimental materials is crucial to minimizing risks of contamination and injury. Additionally, safe disposal of waste is essential to protect both laboratory personnel and the environment from potential hazards.

In this document, we will provide a detailed overview of our team’s approach to laboratory safety regulations, the management of chemicals and biological materials, waste disposal procedures, and the safety education initiatives we have undertaken to promote awareness within our team and the broader community.

In our project, we utilize two well-characterized chassis microorganisms: Escherichia coli and Bacillus subtilis, both of which are extensively studied for their safety and containment.

B. subtilis, a bacterium commonly found in soil, plant surfaces, and even the human body, has a long history of safe use in various applications, such as food and feed additives. Its role in biological control further underscores its safety profile. Extensive research and practical use have confirmed that B. subtilis poses no significant risks to human health or the environment, making it a reliable and safe choice for our project.

Similarly, E. coli, which is part of the Enterobacteriaceae family, has been widely studied and safely used in molecular biology and biotechnology. While certain strains of E. coli can cause illness, the strain we use in our project is a non-pathogenic model organism that has been thoroughly tested for safety in research and industrial applications. We have implemented stringent safety protocols to ensure that this E. coli strain remains contained and does not pose any risk to human health or the environment.

Together, the use of these well-established microorganisms, coupled with strict safety measures, ensures the overall biosafety of our project.

All the genes used in our project have been previously tested in experiments or applied in products, and have been verified as harmless and safe. Specifically, the iaaM and iaaH genes, which are part of the plant-associated IAM pathway, have been utilized in plant-related bacterial systems and have been proven to be safe in prior research (Tsavkelova E., 2012). These genes encode tryptophan monooxygenase and indole-3-acetamide hydrolase, both of which are non-pathogenic and pose no risk to human health or the environment.

Additionally, the fadD gene, included in our project, was previously used by the RDFZ-CHINA team in earlier iGEM competitions and has also been thoroughly tested for safety, demonstrating no adverse effects. This gene, along with other genes in our system, has been verified through extensive literature and experimental data to ensure its non-pathogenic nature.

In our project, we also use other genes related to petroleum degradation and biosurfactant production, such as alkM and laccase, both of which are well-documented in scientific studies as safe and environmentally benign. These genes have been selected not only for their effectiveness in bioremediation but also for their proven safety in various environmental applications.

Though we do not directly employ a typical suicide gene in our system, the proteins expressed by our genetic constructs, including those from our toxin-antitoxin system (such as mazE/mazF), function intracellularly and degrade rapidly in the external environment. If bacterial cell lysis occurs, these proteins will not persist in the environment nor pose a threat to other microorganisms, ensuring that no environmental or ecological harm is caused.

In conclusion, all genes used in our project have been rigorously tested for safety, and the protein products of these genes are non-toxic, ensuring that our project remains safe for both the environment and human health.

We have carefully considered biosafety in our project to prevent any risk of gene leakage. To achieve this, we employed a toxin-antitoxin system (mazE/mazF operon) to create our suicide mechanism.

As an RNA-cleaving enzyme, mazF degrades mRNA, effectively blocking translation and leading to the cessation of bacterial growth, ultimately causing the death of the bacteria. The antidote, mazE, binds to mazF, preventing mRNA degradation and allowing the bacteria to survive in the desired conditions.

Additionally, we utilized PalkB, a special promoter that responds to the presence of alkanes via regulation by the transcription factor AlkS, to control the expression of mazE. In a petroleum-contaminated environment, PalkB is activated, leading to the expression of both mazE and mazF, allowing the bacterial life system to function normally. However, once the petroleum is degraded, or if the bacteria accidentally enter a non-contaminated environment, the lack of alkanes will prevent the expression of mazE, resulting in the natural death of the modified bacteria.

In our project, we ensure laboratory safety through several key aspects, including a biosafety committee, clean-up procedures, proper experimental areas, and comprehensive safety training.

For the biosafety committee, our two research supervisors oversee all laboratory operations, ensuring compliance with safety protocols and preventing operational errors.

Meanwhile, we have implemented strict clean-up procedures. Hazardous materials are disposed of in designated waste bins, which are properly collected and recycled by the laboratory, and classified separately from domestic waste.

In terms of experiment areas, we have established effective isolation measures for all hazardous materials. For example, all experiments involving genetically modified bacteria are conducted in a clean bench, while any work involving toxic materials is performed within a fume hood to ensure safe handling.

Lastly, we have undergone extensive safety training, both prior to and during our laboratory work, covering the following topics:

• Lab access and safety rules

• Hazard identification and safe handling procedures

• Good microbial practices

• Emergency procedures

• Aseptic techniques

• Personal protective equipment (PPE)

Our team also engages in public education on synthetic biology safety. We conduct lectures at school that introduce the usage and safety precautions of synthetic biology products, with a particular emphasis on the safety aspects of our project. In addition, our team attended a round table discussion where we discussed safety concerns and possible resolutions.

To sum up, our team has taken comprehensive measures to ensure biosafety, including the safe use of chassis microbes, safe gene constructs, and the implementation of a complete suicide system. We also prioritize laboratory safety throughout our project development and incorporate biosafety considerations into the design of our products.In addition, we actively promote biosafety education. The combination of these measures ensures our team’s strong biosafety performance.