Laboratory Safety

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1.Laboratory safety management system

Our research strictly complies with China’s relevant biosafety regulations (e.g., the Regulations on Biosafety Management of Pathogenic Microorganism Laboratories and the Regulations on Biosafety Management of Genetically Modified Organisms), as well as applicable national standards. In addition, we enforce the laboratory safety management systems established by Yunnan Normal University and the School of Life Sciences, which include personnel training and evaluation, contained handling of exogenous materials, and autoclave sterilization. All experiments were conducted in laboratories meeting BSL-1 requirements. Team members underwent systematic safety training and performed their work in accordance with iGEM safety and biosecurity policies and laboratory SOPs, thereby ensuring that all research activities were legal, compliant, safe, and well-controlled. Details are as follows:

I. Applicable Chinese National Laws and Regulations

• Regulations on Biosafety Management of Pathogenic Microorganism Laboratories.Defines the standards for the construction and management of biosafety laboratories (BSL-1 to BSL-4)(1).
• Laboratories – General Requirements for Biosafety.A national standard providing systematic requirements for personnel access, protective measures, and waste management(2).
• Regulations on Biosafety Management of Genetically Modified Organisms.Regulates the research, testing, processing, and environmental release of genetically modified organisms(3).
• Measures for the Safety Management of Biotechnology Research and Development.Establishes risk classification and regulatory oversight for scientific research activities involving genetic modification and synthetic biology.

II. Institutional Biosafety Systems at Yunnan Normal University

• Laboratory Safety Management Regulations of Yunnan Normal University:

(1) Cover personnel training requirements, equipment use standards, and laboratory waste management.

(2) All project members must successfully complete safety training and assessments before being granted laboratory access.

(3) Laboratories are equipped with autoclaves as well as facilities for the classified storage of chemicals and biological materials.

These regulations are primarily applied in BSL-1 and plant tissue culture laboratories.

• Laboratory Operating Standards and Registration System of the School of Life Sciences:

(1) Require operation records and approval procedures for microbial experiments, plant transformation, and tissue culture.

(2) Place special emphasis on the contained handling and sterilization of exogenous biological materials such as Agrobacterium rhizogenes and yeast.

(3) Mandate that all experimental materials and liquid waste be documented and sterilized by autoclaving prior to centralized disposal.

III. Safety Guidelines Observed by the Team

The team complies with the iGEM Safety and Security Policy Manual, following all safety policies and compliance checks established for international participants. In addition, we adhere to an internal laboratory operations manual that includes standard operating procedures (SOPs), emergency response plans, and detailed records for the registration and approval of biological materials.

All microbial and plant experiments associated with our project were conducted in laboratories that meet BSL-1 requirements. The team strictly complies with national legislation, institutional regulations, and internal laboratory protocols, and through repeated training ensures that all members maintain appropriate biosafety awareness and response capabilities.

2.Laboratory safety training and management

Strict experimental operation training and safety emergency response training are provided to relevant staff who enter the laboratory to carry out experimental work(Fig 1).

• Laboratory Safety Management Rules of Yunnan Normal University(Fig 2):

(1) Cover personnel entry training, standards for equipment use, and laboratory waste management.

(2) Require all project members to complete and pass safety training and assessment prior to laboratory access.

(3) Laboratories are equipped with autoclaves and facilities for the classified storage of chemicals and biological materials.

• Laboratory Operating Standards and Registration System of the School of Life Sciences:

(1) Implement operation records and approval procedures for microbial experiments, plant transformation, and tissue culture.

(2) Place special emphasis on the contained handling and sterilization of exogenous biological materials such as Agrobacterium rhizogenes and yeast.

(3) Require all experimental materials and liquid waste to be documented and sterilized by autoclaving prior to centralized disposal.

3.Safety Guidelines Observed by the Team

The iGEM Safety and Security Policy Manual: As an international competition team, we comply with all safety policies and compliance checks set by iGEM.The Internal Laboratory Operations Manual: Contains standard operating procedures (SOPs), emergency response protocols, and detailed records for the registration and approval of biological materials.

All microbial and plant experiments in this project were conducted in laboratories meeting BSL-1 requirements. The team strictly adhered to national legislation, institutional regulations, and internal laboratory guidelines, and through repeated training ensured that all members developed the necessary biosafety awareness and response capabilities.

Personal safety protection

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In biological laboratories, personnel safety is a fundamental prerequisite for the successful conduct of scientific research. A comprehensive safety system should encompass risk assessment prior to experiments, standardized procedures during experiments, and emergency response measures for unexpected incidents. Basic protective measures include the use of personal protective equipment (PPE)—such as laboratory coats, gloves, and safety goggles—to prevent injury from biological specimens, hazardous chemicals, or sharp instruments. During experimental operations, aseptic techniques, proper handling of biological materials, and regulated waste disposal procedures must be strictly observed to prevent contamination and cross-infection. Furthermore, particular attention should be directed toward mitigating high-risk events such as aerosol exposure, needlestick injuries, burns, and toxic inhalation. All laboratory personnel are required to undergo systematic safety training and procedural guidance, thereby ensuring that research activities are conducted with safety as the foremost priority and standardized practices as the foundation.

1.Laboratory safety training

Basic laboratory skills training encompasses a range of essential techniques, including plasmid extraction, plasmid cloning, agarose gel electrophoresis, yeast genetic transformation, and product purification. Laboratory safety precautions involve not only maintaining detailed records of instrument usage and accurate labeling of samples, but also strict adherence to waste disposal protocols. To prepare for unforeseen incidents, the college implements rigorous laboratory safety education and training for each cohort of new students and staff, accompanied by evaluations of emergency response capabilities (Fig 3).

Personnel who have not completed safety training or who lack proficiency in basic laboratory skills are not permitted to engage in experimental work. Throughout our laboratory practices, we maintain a rigorous and meticulous approach, ensuring that every detail conforms to established standards. With sustained effort, we aim to achieve laboratory operations that are efficient, safe, and reliable.

2.Personal Safety Training:

Our experiments involve hazardous reagents such as n-hexane, ethyl acetate, methanol, ethidium bromide, and β-mercaptoethanol. Guided by the principle that personal safety is the highest priority, strict protocols are enforced for the handling of such toxic chemicals. To safeguard personnel health and safety, all individuals entering the laboratory must comply with the following personal protective requirements:

• Laboratory coat: A long-sleeved laboratory coat meeting institutional specifications must be worn. The coat should extend to the knees, with cuffs covering the wrists, to effectively prevent contact between hazardous substances and skin or personal clothing. Laboratory coats must remain fastened, clean, and may not be rolled up.
• Trousers and footwear: Long trousers are mandatory. Shorts, skirts, or any clothing leaving skin exposed are prohibited. Footwear must be closed-toe, flat, and fully cover the feet. High heels, sandals, slippers, or other non-protective shoes are strictly forbidden.
• Protective equipment: Depending on the risk level of the procedure, appropriate protective devices must be used, including but not limited to disposable gloves, masks, safety goggles, or face shields. All protective items must be properly worn and disposed of after use.
• Hair management: Hair must be securely tied back to prevent visual obstruction or accidental entanglement during operations.
• Prohibition of accessories: Jewelry such as rings, necklaces, earrings, and bracelets is not permitted, as such items may become entangled with instruments, contaminate materials, or compromise laboratory safety.

3.Post-experiment hygiene and equipment maintenance:

• Hand hygiene: After completing experimental procedures and removing gloves, all personnel must wash their hands thoroughly before leaving the laboratory. If visible contamination is detected during the removal of protective equipment, the process should be paused immediately, hands washed with soap and water or alcohol-based sanitizer, and only then should removal of other protective gear continue.
• Handwashing method: Handwashing with soap and running water for at least 20 seconds is recommended. Alcohol-based sanitizers may be used when conditions are limited, but they cannot substitute for thorough washing when required.
• Clean bench management: Clean benches must be cleaned and disinfected daily according to laboratory protocols. Work surfaces should be wiped with 75% ethanol or other effective disinfectants both before and after use. In addition to routine cleaning, key performance indicators such as airflow velocity and HEPA filter integrity must be evaluated regularly to ensure continuous safe and effective operation. These assessments should be conducted at intervals of no more than six months, with professional maintenance or calibration arranged as needed.

The iGEM laboratory of the School of Life Sciences at Yunnan Normal University has been designated as a BSL-1 facility. It operates in compliance with the Regulations on Biosafety Management of Pathogenic Microorganism Laboratories and the Laboratory Safety Management Regulations for Higher Education Institutions. The laboratory is equipped with the following primary safety facilities(Fig 4):

1. Biological safety cabinets
2. Autoclaves and other sterilization devices
3. Emergency eye-wash stations
4. Mechanical ventilation systems with filtration units
5. Emergency lighting systems
6. Fire-fighting equipment and first aid supplies
7. Flame-retardant, waterproof workbenches resistant to heat, acids, alkalis, disinfectants, and other corrosive chemicals
8. A strict reservation and logging system for laboratory instrument use
9. Emergency showers and decontamination facilitie

Waste disposal methods

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To safeguard the safety of faculty, students, and staff, the laboratory is equipped with dedicated containers for the collection of organic solvent waste. These containers are sealed and stored in designated, well-ventilated areas.

Laboratory waste is strictly segregated by type and hazard level, with proper storage to prevent reactions caused by incompatible wastes. Each waste collection is supervised by designated personnel, with records of the date and type of waste transferred.

Prior to disposal, wastes must be verified as non-toxic and non-hazardous to ensure both environmental and personnel safety. Regular self-inspections are conducted to assess container integrity, including sealing, damage, leakage, labeling, and storage duration.

All containers, infectious substances, and laboratory waste must be clearly labeled and stored in designated areas. Waste is collected centrally and subjected to appropriate physical or chemical treatment according to its characteristics. Waste pending disposal must be kept in dedicated collection boxes with corresponding labels.

Laboratory equipment must undergo regular cleaning, maintenance, and repair. In the event of equipment malfunction, immediate cleaning and disinfection are required to maintain laboratory hygiene and safety.

Potential risks

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1.Laboratory Procedures

I. Experimental organisms

To evaluate the reliability of the REvoDesign platform in protein semi-rational design, we engineered two key enzymes: T5αH, a rate-limiting enzyme in the paclitaxel biosynthetic pathway, and CarRP, an enzyme essential for lycopene biosynthesis. The microbial hosts used were Saccharomyces cerevisiae and Escherichia coli. Following experimentation, all strains were sterilized by autoclaving under the supervision of trained biosafety personnel, thereby ensuring strict biocontainment and environmental safety.

Standard microbial media such as LB and YPD were employed, which contain only the nutrients required for microbial growth. These media are chemically inert and present no known risks to humans or the environment.

Saccharomyces cerevisiae is a non-pathogenic eukaryotic yeast, classified as a Biosafety Level 1 (BSL-1) microorganism, and is widely recognized by regulatory authorities as food-grade safe. It has long been used in fermented food production and industrial enzyme preparation, with no reports of adverse effects on human health or the environment. In synthetic biology applications, the risk of horizontal gene transfer from S. cerevisiae to prokaryotes is negligible, and engineered strains are typically auxotrophic, restricting their survival outside controlled laboratory settings. In this study, physical containment systems (e.g., co-cultivation bioreactors) were combined with biocontainment strategies to further reduce the risk of recombinant strains escaping into the environment.

The E. coli strain DH5α, derived from the K-12 lineage, is a non-pathogenic laboratory strain widely used in education and research. It lacks known virulence factors, pathogenic genes, and antibiotic resistance, and is designated as a BSL-1 microorganism. In addition, it carries mutations that impair its ability to survive in natural environments, precluding environmental persistence or pathogenicity. All manipulations were conducted in accordance with standard microbiological practices, with personnel using appropriate protective equipment. Benches and instruments were disinfected after use, and all cultures and consumables were autoclaved prior to disposal to ensure biosafety and environmental protection.

II. Experimental reagents

All reagents and media employed in this project were subject to rigorous safety evaluation and management. Commonly used media, including LB, YPD, and SD-dropout, are low-risk biological reagents that do not contain toxic chemicals. While they pose no direct hazard to humans, their nutrient-rich composition can support microbial proliferation, presenting a contamination risk. Consequently, all used media and culture waste were autoclaved to ensure complete microbial inactivation prior to disposal, preventing release into the environment.

With respect to organic solvents:

• n-Hexane is a volatile, flammable, and neurotoxic solvent. Prolonged or high-level exposure may lead to neurological symptoms such as headache, dizziness, and sensory impairment. All manipulations were therefore conducted in fume hoods with appropriate protective equipment to prevent dermal and respiratory exposure.
• n-Dodecane has relatively low toxicity but is flammable and may cause mild irritation of the skin and respiratory tract; it was handled with precautions against ignition and inhalation.
• Methanol is the most hazardous solvent used in this project. It is highly toxic and flammable, and ingestion, inhalation, or dermal absorption can cause acute poisoning, including optic nerve damage and potentially fatal outcomes. Its handling followed strict safety protocols, with well-ventilated conditions and mandatory use of gloves and goggles.
• Ethanol is less toxic but remains volatile and flammable. At high concentrations, ethanol vapor can irritate the respiratory tract, and liquid ethanol can irritate the skin and eyes.
• Dimethyl sulfoxide (DMSO) is widely used due to its excellent solubilizing properties but has high dermal permeability, which may facilitate the absorption of other compounds. It was therefore handled with gloves in well-ventilated areas to avoid direct contact.

For antibiotics:

• Kanamycin, an aminoglycoside antibiotic, exhibits moderate toxicity. It may cause skin and respiratory irritation, and long-term exposure carries risks of ototoxicity and nephrotoxicity. Environmentally, it can harm aquatic microorganisms and promote the dissemination of resistance genes.
• Ampicillin, a β-lactam antibiotic, has relatively low toxicity but can provoke severe allergic reactions in individuals sensitive to penicillin. It also contributes to the development of resistant strains in aquatic environments.

All antibiotic-containing waste liquids and culture materials were autoclaved to eliminate antibiotic activity prior to disposal by licensed waste management services, thereby preventing environmental release.

1. Strain activation (Steps 1, 5)
• Risk: Strain instability or contamination during prolonged storage
• Mitigation: Plates stored at 4 °C for more than 7 days were re-streaked for activation. Plates were sealed with Parafilm and labeled with strain code and date before and after activation.
2. Plasmid handling (Steps 2–4)
• Risks: Inhalation of lyophilized plasmid DNA containing antibiotic resistance genes; cross-contamination among transformed colonies
• Mitigation: Plasmid dissolution was performed within a biosafety cabinet. PCR workflows were conducted in physically segregated areas, following the order: template preparation → amplification → electrophoresis.
3. Shake-flask culture (Steps 6–8)

Risk point	Mitigation strategy
Inadequate sealing of deep-well plates	Use gas-permeable sealing membranes (e.g., Breath-Easy®)
Spillage of culture broth	Line trays with absorbent paper during transfers
Condensation during low-temperature induction (20 °C)	Place desiccants inside the shaker

Tab. 2 Risk point and Mitigation strategy

4. Organic phase extraction (Step 9)
• Aqueous phase (containing microbial cells) was immediately sterilized by autoclaving.
• Organic waste was collected in dedicated containers labeled “Flammable Waste – Hexane.”
5. Nitrogen blow-down concentration (Step 10)
• Explosion risk mitigation: nitrogen pressure maintained at ≤ 5 psi; needle positioned > 1 cm above liquid surface; water bath temperature kept ≤ 40 °C (below the boiling point of hexane, 69 °C).
• Loss prevention: samples were transferred into 2 ml screw-cap vials once concentrated to ~1 ml.
• GC-MS analysis (Steps 11–12)
• Risk: Solvent volatilization due to sample vial seal failure
• Mitigation: Pre-slit PTFE/silicone septa caps were used, and sample trays were placed on ice (4 °C) to minimize volatilization.

III. Post-experiment biosafety and waste management

To ensure safety following experimental procedures and the generation of waste, the laboratory implements strict control measures, including systematic personnel training and evaluation, adherence to institutional biosafety regulations, and well-established emergency response protocols.

Tab. 3 Accident types and handling procedures

2. Safety Considerations in Project Design

I. Potential Biosafety Risks

• Horizontal gene transfer and accidental release

Although Saccharomyces cerevisiae poses minimal direct risk to human health, when engineered with exogenous biosynthetic genes (e.g., paclitaxel precursor enzymes), two principal risks arise: (i) accidental environmental release due to operational errors or improper waste management, and (ii) horizontal gene transfer. While gene transfer from yeast to prokaryotes is unlikely, certain conditions (e.g., co-cultivation with bacteria) may still present low-level risk.

• Potential cytotoxicity or bioactivity of metabolites

Paclitaxel precursors such as baccatin III and 10-deacetylbaccatin II are less active than paclitaxel but may still exhibit cytotoxic or bioactive effects. Prolonged or high-level exposure could be harmful to laboratory personnel, and accidental release could affect non-target microorganisms or lower organisms in the environment.

• Use and dissemination of resistance genes

Antibiotic resistance genes are frequently employed as selectable markers in yeast expression vectors. Such genes could, in principle, be transferred to other microorganisms through plasmid transfer or chromosomal integration, thereby contributing to environmental dissemination of resistance. Co-cultivation with bacteria increases the likelihood of such events.

• Improper management of experimental materials and waste

Improper disposal of culture supernatants, lysates, or plasmid DNA could cause laboratory contamination or potential release, underscoring the importance of strict biosafety practices.

II. Safety Measures and Recommendations

• Risk assessment and biosafety classification

Under the Regulations on Biosafety Management of Pathogenic Microorganism Laboratories and international biosafety standards, wild-type yeast is categorized as BSL-1. Engineered strains carrying exogenous elements, however, require reassessment. When constructs involve genetic components with potential ecological consequences (e.g., promoters, terminators, resistance markers), a higher biosafety level may be warranted. It is recommended that biosafety risk assessment reports be submitted to the institutional biosafety committee prior to project initiation, documenting genetic stability and the absence of pathogenic elements.

• lContainment strategies
  • Physical containment: Experiments should be conducted in BSL-2 laboratories with certified biological safety cabinets.
  • Nutritional auxotrophy: Engineering yeast strains with auxotrophic requirements (e.g., histidine or leucine) restricts survival outside laboratory conditions.
  • Genetic safeguards: Incorporating kill-switches or inducible suicide modules enables programmed cell death under defined conditions.
• Operational standards and training

All personnel must complete formal biosafety training and adhere to standard laboratory practices, including the use of protective clothing and aseptic techniques. Designated staff should oversee waste management, ensuring autoclaving or chemical inactivation of all yeast-containing materials.

• Assessment of pathway intermediates

Toxicological evaluation of biosynthetic intermediates, particularly paclitaxel precursors, should be undertaken. Collaboration with pharmacology laboratories may be required for cytotoxicity testing. Any dissemination of results must include disclosure of the potential risks associated with these metabolites.

III. Conclusion

This project is conducted in strict compliance with national and international biosafety and chemical safety regulations, supported by detailed protocols and emergency plans. All reagents and media are handled with appropriate personal protective equipment, and waste is sterilized and professionally collected to minimize risks to human health and the environment.

While yeast itself presents a low biosafety risk, the incorporation of genetic engineering, pathway reconstruction, and production of potentially bioactive compounds necessitates comprehensive risk assessment and preventive measures. Ensuring biosafety is therefore the foundation of responsible innovation and essential for translating laboratory advances into societal benefit.

References

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• 1. Regulations on Biosafety Management of Pathogenic Microorganism Laboratories — https://static.igem.wiki/teams/5915/safety/regulations-on-biosafety-management-of-pathogenic-microorganism-laboratories.pdf
• 2. Laboratories – General Requirements for Biosafety — https://static.igem.wiki/teams/5915/safety/laboratories-general-requirements-for-biosafety.pdf
• 3. Regulations on the Biosafety Management of Genetically Modified Organisms — https://static.igem.wiki/teams/5915/safety/regulations-on-the-biosafety-management-of-genetically-modified-organisms.pdf
• 4. Measures for the Safety Management of Biotechnology Research and Development.
• 5. Yunnan Normal University Laboratory Safety Management Regulations.