Safety

1. Lab Safety

Facility & Access. All work was performed in BSL-1 laboratories suitable for teaching and basic molecular biology. Entry is restricted to trained personnel; visitors require supervisor approval and must be accompanied at all times.

Training & Documentation. Every team member completed lab safety training before bench work (chemical hygiene, biosafety, biological waste, fire safety, electrical safety, emergency response). Equipment-specific SOPs (centrifuge, autoclave, lyophilizer, gel apparatus, shaker incubator, biosafety cabinet) were read and signed. Experiments are recorded in bound notebooks or ELN with dates, lot numbers, and responsible person.

PPE & Hygiene. Lab coat (closed), nitrile gloves, goggles/face shield, long pants, closed-toe shoes are mandatory. No food/drinks/cosmetics. Gloves are changed after chemical handling, before touching shared surfaces, and between GMO/non-GMO workflows.

Work Practices & Engineering Controls.

Biosafety cabinet (Class II) is used for GMO manipulations (plating, inoculation, transformation) and Agrobacterium rhizogenes operations. Surfaces are decontaminated with 70% ethanol and final 10% bleach contact ≥10 min.

Sharp safety: use safety scalpels where possible; dispose in approved sharps containers.

Centrifuge safety: balance within 0.01–0.05 g; inspect tubes/rotors (no cracks); never open until full stop.

Autoclave: biological waste at 121 °C, 15 psi, ≥30 min; verify autoclave tape/indicator; cool before disposal.

UV/Blue-light imaging: use shields/eyewear; avoid direct UV exposure.

Lyophilizer & grinding: prevent aerosol/powder exposure; grind plant powder in a fume hood or powder hood; wear dust mask/respirator if needed.

Shaker incubators: secure flasks; keep doors closed during operation.

Chemical Safety—project-specific reagents. Handle all in a fume hood, label clearly（name/concentration/date/owner/hazards）.

Antibiotics（kanamycin, rifampicin, streptomycin, chloramphenicol）: avoid skin contact and inhalation; minimize spills; inactivate culture waste before disposal（bleach ≥10%, ≥30 min）; do not pour active antibiotics down the drain.

β-estradiol（endocrine-active）: prepare stocks in DMSO inside hood, wear double gloves; store light-protected; collect residues as hazardous chemical waste.

Syringic acid & 4-guanidinobutanol（substrates）: weigh in hood; avoid inhalation; clean spills immediately with absorbent and detergent, then bleach if GMO is present.

UDPG（nucleotide-sugar）: low hazard but treat as chemical; avoid skin/eye contact.

Ni-NTA resins & imidazole（protein purification）: Nickel is a heavy metal—all resin and imidazole-containing eluates/washes go to heavy-metal waste; wear goggles and double gloves.

Methanol（70% extraction; flammable）: store in flammables cabinet; keep away from ignition sources; waste to organic solvent waste only.

Electrophoresis buffers & stains: use non-mutagenic dyes; if UV-excitation dyes are used, treat gels and rinses as hazardous waste per institutional policy.

Waste Segregation & Disposal.

Biohazard: cultures, GMO plant tissues, tips—autoclave, then red-bag disposal per institutional SOP.

Chemical: methanol/DMSO/imidazole/β-estradiol solutions—collect in clearly labeled waste bottles; no drain disposal.

Heavy metal: Ni-NTA resin, nickel-containing eluates—dedicated container for pickup.

Mixed waste: follow the stricter category; when in doubt, treat as chemical hazardous waste.

Spill & Incident Response.

Biological spills: cover with towels, apply 10% bleach (≥10 min), then wipe with 70% ethanol.

Chemical spills: use spill kit (absorbent/neutralizer), ventilate, wear PPE; for flammables (methanol) remove ignition sources; notify supervisor.

Exposure/accidents: eye wash ≥15 min, safety shower for skin; report to supervisor and complete incident form.

Storage, Transport & Labeling.

All cultures/plates/tubes labeled with content, date, owner, hazards.

Transport cultures within secondary containment (sealed boxes) inside the building only; no off-site transport.

Antibiotics/β-estradiol/solvents in locked chemical cabinets; GMO materials in dedicated cold storage; logbook for plasmids and strains.

2. Project Safety

The chassis strains used include Escherichia coli DH5α for plasmid construction and E. coli BL21(DE3) for protein expression, both classified as non-pathogenic laboratory strains under BSL-1 standards. For plant experiments, Nicotiana tabacum hairy roots were induced using Agrobacterium rhizogenes Ar.1193, which is also widely used as a standard tool in plant biotechnology.

The plasmids employed are pRSFDuet-SUMO and pETDuet-MBP, both low-risk vectors designed for protein expression, without toxins, pathogenic genes, or antibiotic-resistance markers beyond routine lab use. The plant vector pAGM4723 is part of the MoClo system and carries only standard selectable markers.

3. Product Safety

The target product leonurine is a natural alkaloid traditionally derived from Leonurus japonicus. It has long been used in traditional Chinese medicine and exhibits low toxicity at therapeutic concentrations. Our project does not involve mammalian cell experiments, but risk is controlled through:

​ ✓ Concentration control: yields in our experiments are at microgram levels, far below toxic thresholds.

​ ✓ Environmental degradation: leonurine is an organic compound that can be degraded by natural microbial and enzymatic processes.

​ ✓ Intended use: the final product is envisioned as an active pharmaceutical ingredient (API) supplied to pharmaceutical companies, not directly released into the environment or consumer products during iGEM experiments.

4. Environmental & Ethical Safety

All genetically modified organisms (GMOs) were handled strictly within closed laboratory conditions. No GMOs were released into the environment. Biological waste, including bacterial cultures and plant tissues, was autoclaved or treated with disinfectants before disposal. Chemical waste was segregated and disposed of following institutional regulations.

From an ethical perspective, our project focuses on developing a safer and cost-effective route to an existing natural compound, without creating new risks. Human Practices interviews were conducted with expert consent, and no personal sensitive data was collected.

5. Compliance & Responsibility

All work complied with iGEM’s safety and security rules, as well as national biosafety regulations. As a high school iGEM team, we strictly limited our experiments to BSL-1 organisms and approved plant systems. No work was performed with pathogenic strains, mammalian cells, or toxic genes. Future applications of our system will involve standard pharmaceutical safety evaluations, including preclinical toxicity testing, before any clinical or industrial use.