Contents

    Laboratory Safety

    1. Daily Safety in BSL-2 Laboratories

    Safety is the core principle of our project, which runs through every link, from gene cloning, strain culture, fermentation optimization, product extraction to the final use of toxins. Our research focuses on using synthetic biology technology to construct genetically engineered strains that highly produce mycotoxin-degrading enzymes and optimizing their fermentation processes.

    Considering the chassis organisms we use---Pichia pastoris X-33, Escherichia coli (E. coli) DH5α, and E. coli B strain BL21---as well as various chemicals involved in the experiments, such as methanol, acetonitrile, hydrochloric acid solution, aflatoxin, ochratoxin, patulin, and zearalenone, all experiments are conducted in a laboratory environment at Biosafety Level 2 (BSL-2), and strictly comply with national biosafety regulations and iGEM safety guidelines.

    We have formulated a detailed safety management system to ensure that every step of the experimental operation is safe and controllable.

    Classroom discussion

    2. Biosafety and Containment

    Our project involves a variety of biological agents that require careful handling.

    2.1 Handling of Microorganisms

    We mainly use E. coli (DH5α for cloning and BL21 for protein expression) and Pichia pastoris (X-33) as expression hosts. These strains themselves are classified as Risk Group 1 and are non-pathogenic.

    All constructed engineered strains are stored as glycerol stocks in a dedicated -80°C refrigerator. Each strain has a clear label indicating the strain name, genotype, construction date, and person in charge. When glycerol stocks are taken out of the refrigerator for resuscitation, the operation must be carried out in a biosafety cabinet. Only a small amount of bacterial solution is taken for streaking or inoculation to avoid damage to the strains caused by repeated freezing and thawing.

    All liquid cultures are conducted in sealed shake flasks, with the flask mouths covered with breathable sterile sealing film. This not only ensures ventilation but also effectively prevents strain leakage and cross-contamination. The fermentation process of Pichia pastoris is carried out in a dedicated fermenter, and the exhaust pipeline of the fermenter is equipped with a high-efficiency air filter to ensure that the exhausted gas is filtered and prevent the aerosol release of engineered bacteria.

    2.2 Genetic Modification and Potential Risks

    We introduce exogenous genes ADH3 and LlADH, which can degrade mycotoxins, into these chassis organisms through genetic engineering methods. Although the function of these enzymes is to degrade toxins, the cultivation and handling of engineered strains must still be carried out in accordance with BSL-2 standards to prevent any potential and unforeseen risks.

    2.3 Containment Strategies

    All operations involving live genetically engineered bacteria are performed in a Class II Biosafety Cabinet (BSC). Biosafety cabinets undergo regular maintenance, certification, and disinfection. We implement a strict laboratory access system---only team members who have received safety training and obtained authorization can enter the laboratory. The laboratory maintains good ventilation, adopts a negative-pressure safety mode, and is equipped with HEPA air filters to prevent aerosol diffusion.

    Classroom discussion

    3. Chemical Safety

    Our experiments involve a variety of chemicals, some of which have specific hazards and require special handling.

    3.1 Main Chemicals and Their Risks

    • Antibiotics: During strain screening and culture, we use antibiotics such as ampicillin to maintain selective pressure. These antibiotics may cause allergic reactions. When weighing the powder, dust masks and gloves must be worn to avoid inhalation.
    • IPTG: As an inducer for the T7 promoter in E. coli, it is somewhat irritating. Gloves must be worn during operation to avoid direct skin contact.
    • Methanol: In the induced expression stage of Pichia pastoris, we use methanol as a carbon source and inducer. Methanol is flammable and toxic. All operations must be carried out in a fume hood and away from any sources of ignition.
    • Mycotoxin Standards: In enzyme activity detection experiments, we use trace amounts of ochratoxin A (OTA) and aflatoxin B standards. These substances are highly toxic and carcinogenic. Weighing and dilution must be carried out in a biosafety cabinet. During operation, double gloves and a protective face shield must be worn strictly, and disposable consumables must be used to prevent any form of contact.
    Classroom discussion

    3.2 Personal Protective Equipment (PPE)

    All personnel entering the laboratory must wear appropriate personal protective equipment, regardless of the type of operation. According to the different operation risks, we divide the protective equipment into two levels: basic protection and enhanced protection.

    3.2.1 Basic Protective Equipment

    This is the minimum protective requirement for all experimental operations, applicable to routine operations that do not involve direct contact with high-risk substances, such as balancing, cleaning, or streaking of non-pathogenic strains on ordinary experimental benches.

    • Lab Coat: A long-sleeved lab coat that covers the entire body must be worn, and the material should be waterproof or splash-proof. The lab coat should be kept clean and tidy, and the cuffs must be fastened. Wearing a white coat or other outerwear over the lab coat is prohibited. The lab coat must not be worn outside the laboratory.
    • Safety Glasses: Standard-compliant safety glasses or goggles must be worn to protect the eyes from chemical splashes, aerosols, or broken glass. Contact lenses cannot replace safety glasses.
    • Gloves: Disposable nitrile gloves must be worn. Choose gloves of the appropriate size to ensure a good fit on the hands. Check for damage before putting on the gloves. After handling different samples or completing an operation step, the gloves must be replaced in a timely manner to prevent cross-contamination.
    • Shoes: Shoes that cover the entire foot, such as sports shoes or leather shoes, must be worn. Wearing slippers, sandals, or open-toe shoes is prohibited to prevent foot injuries caused by chemical spills or falling sharp objects.
    • Others: Personnel with long hair must tie their hair up. Wearing jewelry that may affect operations or cause contamination, such as long necklaces, bracelets, and rings, is prohibited.

    3.2.2 Enhanced Protective Equipment

    When conducting high-risk operations, the following additional protective equipment must be added on the basis of basic protection.

    • Double Gloves: When handling toxic substances (such as OTA and aflatoxin standards) or performing high-risk operations such as fermenter inoculation, double nitrile gloves must be worn. If the outer gloves are damaged or contaminated during operation, they should be replaced immediately, and the inner gloves serve as backup protection.
    • Protective Face Shield: When handling highly volatile or highly toxic substances such as mycotoxin standards and methanol, a protective face shield must be worn. The face shield should fully cover the nose and mouth area to provide respiratory protection and prevent inhalation of toxic aerosols or vapors.
    • Chemical Protective Apron: When transferring large quantities of chemicals, preparing strongly corrosive solutions, or handling mycotoxins, a waterproof and chemical-resistant apron must be worn over the lab coat to protect the torso and legs.
    • Mask: When weighing powders that are prone to generating dust, such as antibiotic powders and talcum powder, an N95 or higher level dust mask must be worn to prevent inhalation of the powder.
    • Special Shoe Covers: When performing operations inside the fermenter or handling experiments that may cause severe contamination, such as large quantities of bacterial solution, disposable waterproof shoe covers must be worn over the shoes.

    4. Waste Management

    To prevent environmental pollution and personal injury, we have established a clearly classified waste disposal process.

    4.1 Biological Waste Disposal

    • Solid Biological Waste: All consumables that have come into contact with engineered bacteria, such as petri dishes, pipette tips, centrifuge tubes, and gloves, must be placed in dedicated biological waste bags. After sealing, they are treated by high-pressure steam sterilization at 121°C for 30 minutes. Only after confirming sterility can they be discarded as ordinary garbage.
    • Liquid Biological Waste: For liquid waste such as fermentation broth and bacterial solution, sodium hypochlorite (84 disinfectant) with a final concentration of 10% must be added first. After thorough mixing, it is allowed to stand for at least 1 hour for inactivation treatment before being discharged into the dedicated sewer of the laboratory.

    4.2 Chemical Waste Disposal

    • Organic Solvents: Waste organic solvents such as methanol and ethanol must be collected in dedicated sealed containers with clear labels and regularly recycled and treated by qualified professional companies.
    • Mycotoxin Waste: All consumables (including tips, centrifuge tubes, and reaction plates) and solutions that have come into contact with mycotoxins must be soaked in a 5% sodium hypochlorite solution for more than 24 hours before being autoclaved or discarded.
    • Sharp Waste: Needles, broken glassware, etc., must be placed in dedicated puncture-resistant sharps containers.

    5. Equipment Safety

    We have formulated strict operating specifications for the professional equipment used.

    • Biosafety Cabinet: Before and after each use, the workbench surface must be wiped and disinfected with 70% ethanol. The UV lamp and HEPA filter should be replaced regularly, and maintenance records should be kept properly.
    • Autoclave: Only trained personnel are allowed to operate it. The loaded items should not exceed 80% of the capacity to ensure that cold air can be completely discharged. After sterilization, the pressure and temperature must be reduced to the safe range completely before opening.
    • Fermenter: Before use, check whether all pipeline connections are tight and free of leaks. During operation, closely monitor parameters such as temperature, pH, and dissolved oxygen. When adding methanol, ensure that the pipeline is unobstructed and the operation is carried out in a well-ventilated environment.
    • Centrifuge: Before centrifugation, ensure that the sample tubes are strictly balanced. Use sealed centrifuge tubes or centrifuge cups to prevent sample leakage and aerosol generation.

    6. Safety Training and Emergency Preparedness

    • Comprehensive Safety Training: Before entering the laboratory, all team members receive safety courses. All team members must complete BSL-2 laboratory safety training and pass theoretical and operational exams. The training content includes biosafety, chemical safety, fire and explosion prevention, and emergency handling.
    • Regular Safety Meetings: We hold a safety meeting every month to review the safety situation in recent experiments, discuss potential risks, and update safety operation specifications.

    Emergency Plans

    • Chemical Spill: In case of methanol spill, immediately turn on ventilation, cover the spilled area with absorbent cotton, and then rinse with a large amount of water.
    • Mycotoxin Exposure: In case of accidental skin contact, rinse immediately with a large amount of water and soap. If it splashes into the eyes, rinse with an eye washer for at least 15 minutes immediately and seek medical help promptly.
    Classroom discussion
    • Fire: In case of a small-scale fire, use an appropriate fire extinguisher (such as a dry powder fire extinguisher) to put it out. If the fire is out of control, evacuate immediately and call the police.