1. General Principles
Throughout the entire project cycle, this project strictly adheres to the iGEM Safety Policy and the biosafety management system of the host institution. All experimental activities are conducted in a BSL-1 laboratory, with no human or vertebrate experiments, environmental release, or field trials performed. We only use standard, non-pathogenic microbial chassis (E. coli K-12 derived strains and BL21(DE3)) as well as common metabolic enzyme genes (GPS, CsTPS1, GeDH, and their rational mutants), and do not involve toxins, virulence factors, antibiotic-modifying enzymes, or genes related to increased pathogenicity.
2. Biological Materials and Genetic Elements
The chassis bacteria are E. coli K-12/BL21(DE3), which are non-pathogenic strains commonly used in teaching and industry. The expression vectors used are pET-21a(+) and pBAD23, both of which are non-conjugative and non-broad-host-range plasmids, containing only the conventional selection marker Amp^R. The target genes are coding sequences of metabolic enzymes, derived from plant/microbial pathways or their codon-optimized versions; we have confirmed through bioinformatics screening that they do not contain toxins, elements related to antibiotic resistance transmission, key factors of phage lysogenic cycles, or CRISPR interference modules. All synthetic DNA and bacterial strains entering or exiting the laboratory are registered and stored in the inventory, and are subject to Material Transfer Agreements (MTA) and import/export and transportation regulations.
3. Biosafety and Operational Specifications
Routine operations, including cloning, transformation, shake flask culture, qPCR, Western Blot, and HPLC-UV detection, are all carried out under BSL-1/2 conditions. Bacterial cultures, bacteria-containing consumables, and agar plates are autoclaved before leaving the experimental bench and then disposed of as biohazardous waste. Prior to the start of experiments, experimental procedures and protocols are reviewed; all operations are performed with standard Personal Protective Equipment (PPE), including lab coats, disposable gloves, and goggles/face shields, and work is conducted near a spill response kit. Steps that generate aerosols (such as vigorous vortexing, opening centrifuge tubes, and colony picking) are completed in a biosafety cabinet. All culture media, cell materials, and plasmids are used exclusively within the laboratory, are not removed from the experimental area, and are not discarded through drains or regular trash cans.
4. Chemical Safety and Management of Volatile Monoterpenes
Common reagents such as IPTG, L-arabinose, Tris, SDS, and transfection reagents are handled in accordance with their respective Safety Data Sheets (SDS). Geraniol and citronellal are volatile monoterpenes/aldehydes, which are irritating and flammable; all preparation, uncapping, and extraction processes are conducted in a chemical fume hood, and volatile organic solvents are not handled in biosafety cabinets. Acetonitrile/methanol used in HPLC is stored and discarded in compliance with organic solvent regulations (with separate disposal of halogenated and non-halogenated solvents). Waste liquids and cleaning solutions containing monoterpenes/aldehydes are collected separately and disposed of by the university's hazardous chemical disposal system. When working with flammable solvents, keep them away from open flames and heat sources, ensure electrostatic grounding, and tightly seal containers immediately after use. In case of skin/eye contact, rinse with plenty of running water immediately, and record and report the incident.
5. Product and Exposure Assessment
The project products are low-molecular-weight monoterpene derivatives such as citronellal, which are targeted as candidate ingredients for "green mosquito repellents". Only microscale synthesis and analysis are conducted in the laboratory, with no human trials or environmental diffusion experiments performed. Considering the risk of skin and respiratory tract irritation from monoterpenes/aldehydes, we limit the maximum allowable indoor operation volume, use sealed centrifuge tubes, store samples at low temperatures, and transport them in secondary containers; when necessary, disposable activated carbon masks are worn to reduce inhalation exposure. In the event of a spill, first absorb the spilled material completely with an inert adsorbent, then wipe the area with 75% ethanol/cleaner, and dispose of the waste as hazardous chemicals.
6. Waste Disposal and Decontamination
Biological waste (including bacterial cultures, plates, and pipette tips) is subjected to moist heat sterilization at 121 °C for 30 minutes, labeled as "autoclaved", and then placed in dedicated biohazard bins; sharp objects are placed in puncture-resistant sharps containers. Chemical waste is classified into three categories: organic solvents, aldehyde/monoterpene-containing solutions, and general experimental chemical waste, which are placed in corresponding labeled containers and not mixed with biological waste. Decontamination is performed using 75% ethanol and 1% sodium hypochlorite (selected based on material compatibility); instrument surfaces are wiped down before the end of each workday. Clean benches/biosafety cabinets undergo routine UV disinfection and surface disinfection procedures, which are documented.
7. Personnel Training and Access
All team members complete biosafety and chemical safety training for the laboratory before joining the team, and can only operate independently after passing the assessment; specialized training for equipment such as biosafety cabinets, centrifuges, and HPLC is recorded separately. New members must complete their first three key operations (transformation, aseptic transfer, and solvent loading for instrument analysis) under the supervision of a supervisor or teaching assistant. The accident reporting mechanism is publicly available: any spills, needle sticks, chemical exposures, equipment malfunctions, or non-compliance with procedures must be documented in an incident form within 24 hours, and the safety officer will review and verify improvement measures.
8. Transportation and Storage
Plasmids, bacterial strains, and standard samples are all sealed in secondary containers, attached with hazardous material labels and SDS summaries, and transported on campus via designated routes and during specified time windows; external cooperation or shipment strictly follows channels with qualified carriers. Frozen inventory is backed up twice at -80 °C, and the consistency between labels and electronic records is verified regularly; expired samples are uniformly discarded after autoclaving.
9. Environment and Ethics
This project does not conduct any form of environmental release, field trials, or capture of wild vector insects; if entomological assessment is required, it will be limited to pathogen-free, long-term laboratory-reared strains in a qualified entomological laboratory, and will comply with the ethics and safety regulations of the corresponding institution. We do not collect human samples or conduct volunteer exposure tests or skin patch tests.
10. Risk Assessment and Mitigation
At the pathway level, the main risks are the stress of intermediates/products on cells and the escape of volatile substances. We achieve flux balance through dual induction and hierarchical expression, prioritize ensuring that the intensity of the terminal GeDH is not lower than that of the upstream, and if necessary, adopt in-situ encapsulation/microextraction to reduce the peak concentration in the aqueous phase. At the experimental level, the main risks are the volatility and flammability of solvents and monoterpenes, which have been controlled through engineering measures such as fume hoods, low dosages, sealed transportation, and classified disposal. At the organizational level, we have established a closed loop of pre-training, in-process inspections, and post-event reviews to ensure that "risks are identifiable, measures are implementable, and records are traceable".