Safety is a fundamental priority throughout our project, which focuses on the preparation of microbial media, cultivation of Escherichia coli, construction and verification of expression vectors, introduction of vectors into competent cells, and related protein testing experiments. Our research involves microbial culture, molecular cloning, protein purification, and other operations, all of which require strict adherence to safety protocols to protect researchers, the environment, and public health. This section details the safety measures implemented in our project, covering project-specific safety considerations and general laboratory safety practices, in line with relevant safety guidelines.

Fig .1 Safety notice before entering the lab zone

Our project primarily uses Escherichia coli strains (including those for expression vector production and BL21 competent cells for protein expression). These strains are non-pathogenic and low-risk, classified as Biosafety Level 1 (BSL-1) organisms. Key safety measures include:

Sterilization: All microbial media (LB liquid and solid media) and contaminated materials (e.g., pipette tips, centrifugal tubes used in bacterial culture) are sterilized by autoclaving at 121°C for 20 minutes to eliminate viable organisms before disposal.

Containment: Bacterial activation culture, colony picking and other operations involving live bacteria are performed in a super-clean bench to prevent aerosol spread and cross-contamination.

Strain Management: Strains are clearly labeled and stored appropriately. No strains with enhanced virulence or unnecessary antibiotic resistance are constructed.

A variety of reagents are used in experiments, with specific safety measures based on their properties. The hazardous chemical reagents used in our laboratory are managed, stored and authorized for use by a unified administrator. The reagents are uniformly stored in the reagent safety cabinets.

Fig .2 Safety Cabinet for the storage of chemical regent

Our experimental environment is managed to minimize risks during operations:

Equipment Safety:

• Centrifuges are operated with balanced loads, and tubes are checked for cracks to prevent leakage.
• Ultrasonic Cell Disruption Machines are used with ice baths to control temperature, reducing heat-induced protein damage and ensuring operator safety.
• Electrophoresis equipment is inspected for electrical safety; operators dry hands before handling to avoid electric shock.

Contamination Control:

• Work surfaces, especially in super-clean benches, which are disinfected with appropriate agents before and after experiments.
• UV lights in clean benches are used only when unoccupied to prevent eye and skin damage, with clear warnings.

Waste Management:

• Biological waste (e.g., bacterial cultures, contaminated pipette tips) is autoclaved before disposal.
• Chemical waste (e.g., dye-containing gels, leftover imidazole solutions) is segregated into labeled containers and disposed of following laboratory protocols.

Fig .3 Experimental waste and household waste are handled separately.

5.1 Legal Compliance

Our project strictly adheres to national and institutional regulations regarding laboratory biosafety, chemical management, and waste disposal. All operations involving microorganisms, reagents, and equipment comply with relevant guidelines for biological and chemical safety, ensuring no violations of legal requirements.

5.2 Training

All team members received mandatory safety training before conducting experiments:

Basic Safety Training: Covered hazard identification of reagents and equipment, as well as proper use of personal protective equipment (PPE).

Task-Specific Training: Hands-on training for key operations, such as autoclaving media, plasmid extraction, and electrophoresis, to ensure proficiency and safety.

5.3 Laboratory Rules

To maintain a safe working environment, the following rules are strictly enforced:

1. PPE Requirement: Lab coats, gloves, and appropriate eye protection are mandatory when handling reagents, conducting microbial experiments, or operating equipment.
2. Sterile Technique: Strict aseptic procedures are followed during media preparation, bacterial culture, and transformation to prevent contamination.
3. Labeling: All tubes, plates, and reagents are clearly labeled with content, date, and operator to avoid confusion.
4. Prohibited Actions: No eating, drinking, in the laboratory; no unauthorized modification of experimental protocols or equipment.
5. Emergency Response: Spills (e.g., chemical or bacterial culture spills) are immediately contained and cleaned up following established procedures; incidents are reported to supervisors promptly.
6. By implementing these comprehensive safety measures, we ensure that all experiments are conducted safely, protecting the well-being of researchers and the environment.

Fig. 4 Emergency shower device and first aid kit