• Safety First: The team places a strong emphasis on safety, offering both online and in-person training on laboratory safety practices, including proper pipette handling and cell culturing techniques. Adhering strictly to the iGEM Safety Policies and internal protocols, they ensure their research remains confined to the relatively low-risk bacterium E. coli.
• Biosafety Committee Oversight: A dedicated biosafety committee, comprising research faculty, oversees laboratory practices to ensure proper disposal, sanitation, and safety.
• Comprehensive Training: Team members receive training on various aspects of biosafety, including biosafety levels, equipment usage, good microbiological practices, emergency procedures, and data biosecurity.
• Risk Mitigation: The document delineates various risk management tools, including accident reporting procedures, requirements for personal protective equipment, and specific laboratory regulations such as designated waste disposal and mandatory handwashing.
• Zero Accidents: The team underscores the effectiveness of their safety protocols by reporting no accidents during their experiments.

L-5-Methyltetrahydrofolate (L-5-MTHF) is the primary form of folate in serum and the only biologically active form in humans. It is also the only folate capable of crossing the blood-brain barrier. As the bioactive form of folic acid, L-5-MTHF is crucial for nucleic acid synthesis, cell growth, tissue repair, and embryonic development (1-4). Humans are unable to synthesize folic acid and must obtain it from dietary sources. In the body, folic acid is reduced to tetrahydrofolate, the coenzyme for one-carbon transferase, through the action of dihydrofolate reductase. Tetrahydrofolate then binds to a one-carbon unit and, via a series of enzymatic reactions, is converted into the biologically active form L-5-MTHF (5). The compound can be commercially supplied to individuals with impaired folic acid metabolism. However, current commercial production primarily depends on chemical synthesis (6-9). This presents challenges such as limited supply, safety concerns, and environmental risks (10).

Before initiating any experiments, the team underwent comprehensive online safety training, which covered pipette use and cell culturing. This training was concluded with a quiz to verify understanding. The team strictly followed iGEM Safety Policies by exclusively using the relatively safe bacterium E. coli and ensuring its containment to prevent environmental release. Additionally, the biosafety committee provided offline educational sessions.

The biosafety committee, comprising two research faculty members, is responsible for overseeing all aspects of laboratory safety. The committee conducts regular inspections to ensure proper waste disposal, sanitation, and overall safety in the work areas. Additionally, it provides personalized safety training to each team member.

The training encompassed a wide array of topics, including:

• Lab access rules and protocols
• Roles and responsibilities of individuals
• Understanding different biosafety levels
• Proper use of biosafety equipment (e.g., biosafety cabinets)
• Good microbial techniques and lab practices
• Disinfection and sterilisation procedures
• Emergency protocols
• Rules for transporting materials
• Physical and personnel biosecurity
• Dual-use research and experiments of concern
• Data biosecurity
• Chemical, fire, and electrical safety

The primary work area was the open bench.

The team worked with the following strains of Escherichia coli: DH5α and BL21 (DE3).

Several risk management tools were in place, including:

• A detailed accident reporting procedure with emergency contact information.
• Mandatory use of personal protective equipment (PPE) such as lab coats, gloves, and eye protection.

General safety regulations included:

• Prohibition of running or sudden movements.
• No food or drinks allowed in the lab.
• Strict waste disposal protocols, with designated bins and sterilisation procedures.
• Mandatory sanitation of pipettes after each use.
• Adherence to user manuals for all equipment.
• Required use of gloves and lab coats during experiments.
• Handwashing upon entry and exit and appropriate attire.