Safety

Preface

  Safety has always been the core priority throughout the entire process from the start to the completion of this project, covering two main aspects: one is the design safety of the engineered biological system itself, and the other is the safety of laboratory operations during experimental operations. The team has implemented strict security measures to mitigate potential risks and ensure full compliance with relevant regulations and standards.

Design security

  Our project focuses on the pollution control of polycyclic aromatic hydrocarbons (PAHs) and constructs a naphthalene-phenanthrene co-degradation system through synthetic biology technology: the nah gene cluster responsible for naphthalene degradation, the phnC gene cluster degraded by phenanthrene, and the rhamnolipid synthesis gene rhl, which enhances the water solubility of PAHs, are co-heterologous expressed in engineered Escherichia coli to achieve efficient degradation. To ensure the safety of the design of engineered strains, the team carefully screened the genetic elements and tightly controlled their function – neither enhancing the pathogenicity of the microorganism nor introducing any unintended harmful properties.

2.1 Screening and source of gene elements

  The genetic elements integrated into engineered Escherichia coli are derived from well-characterized microbial strains and are limited to promoting PAHs degradation without additional risk.

  Rhamnolipid synthesis gene (rhl): This gene is derived from Pseudomonas aeruginosa and works to promote bacterial secretion of rhamnolipids. Rhamnolipids can improve the water solubility of polycyclic aromatic hydrocarbons, thereby improving the absorption and degradation efficiency of PAHs by engineering bacteria. The study only cloned the rhl gene and heterologous expression in Escherichia coli, without any genetic modification of the original Pseudomonas aeruginosa strain, ensuring that the virulence of Pseudomonas aeruginosa would not be enhanced.

  Polycyclic aromatic hydrocarbon degradation gene clusters: the nah gene cluster responsible for degrading naphthalene and the phnC gene cluster responsible for degrading phenanthrene are both derived from Pseudomonas putida. With the help of these gene clusters, engineered Escherichia coli is able to absorb typical PAHs such as phenylphenyl from the environment, internalize them, and metabolize them into harmless products.

2.2 Safety assessment of donor strains

  The team conducted a comprehensive safety assessment of the strains that provided key genes to ensure that their use did not introduce unnecessary risks.

  Pseudomonas aeruginosa strain selection: Although the Pseudomonas aeruginosa PA14 strain is slightly more pathogenic than the PAO1 strain, we chose this strain because the laboratory already maintains its strain. Furthermore, studies have only used PA14 as a PCR template only which involved limited conduction with live bacteria. In addition, While other bacteria such as Pseudomonas putida, Pseudomonas fluorescens, and Burkholderia spp. possess analogous genes or produce similar compounds, Pseudomonas aeruginosa remains the most efficient and extensively studied producer of rhamnolipids to date, and the risk to human health is lower under the premise of standardized operation.

  Safety of other donor strains: Pseudomonas putida, which provides polycyclic aromatic hydrocarbon degradation gene clusters is classified as a GRAS (Generally Recognized as Safe) strain that has been widely used in environmental remediation research for a long time.

Laboratory safety

3.1 Personnel training and emergency preparedness

  Training requirements: All team members have completed Biosafety Level 2 (BSL-2) training and project-specific safety training, including: laboratory access rules (e.g., dress code, no eating and drinking); standardized microbial operation technology, disinfection and sterilization process; Emergency response procedures (for exposure or leakage events); Knowledge of chemistry (toxic/flammable) and electrical safety, etc.

  Emergency Plans: Develop detailed emergency response plans to deal with accidents quickly and get immediate consultation assistance.

  Supervision and inspection: supported by biological laboratories and supervised by laboratory safety committees. Relevant personnel will regularly carry out safety inspections and guidance, and promptly supervise and rectify irregular operations when they are found.

3.2 Laboratory facility compliance and operation control

  Biosafety level compliance: All experiments involving Pseudomonas aeruginosa were conducted within the BSL-2 laboratory; Bacterial culture, gene cloning, and other operations are completed in a secondary B2 type biosafety cabinet (BSC) to prevent aerosol leakage. Experiments involving the toxic chemical phenanthrene are carried out in fume hoods to avoid inhalation or skin contact.

  Personnel Protection: Experimenters should wear a full set of PPE, including disposable gloves, lab coats, face shields, and goggles, when performing high-risk operations. The integrity of PPE should be checked before use, and disposed of according to regulations after use.

  Strict waste management: All materials contaminated by Pseudomonas aeruginosa and waste generated by engineering bacteria experiments must be autoclaved to inactivate pathogens, and then packed into special containers with leak-proof and clearly marked labels, and transferred to professional institutions for disposal according to compliance procedures. It is strictly forbidden to mix hazardous waste with ordinary household waste and dispose of it.