Lab Safety ​

At HKUST (Guangzhou), our iGEM team recognizes that laboratory safety is the foundation of all wet lab work. We have established strict safety protocols to ensure that all experimental activities are conducted responsibly, protecting both our team members and the wider community.

Safety Training and Certification ​

• Mandatory Safety Exams: All wet lab members successfully completed the official HKUST(GZ) laboratory safety exams, covering:
  1. General Laboratory Safety
  2. Chemical Safety I – Chemical Safety for Laboratory Users
  3. Chemical Safety II – Hazardous Waste Management
  4. Biological Safety
  5. Laser Safety
  6. Pressure Safety
  7. Respiratory Protection
  8. Electrical Safety
• Member Access Control: Only certified wet lab members are permitted to conduct experiments. Non-wet lab members have signed an official guarantee with our PI ensuring they do not enter the laboratory for experimental purposes.

Additional Hands-On Training ​

• Before starting experiments, all wet lab members received an extra week of hands-on training led by our PIs and advisors. This training emphasized:
  • Standard laboratory operating procedures
  • Emergency response measures (e.g., chemical spills, fire, biological exposure)
  • Proper use of personal protective equipment and safe waste disposal
• Instrument-Specific Training: Members must complete specialized training before using large-scale equipment. No member is allowed to operate an instrument without prior authorization.

Laboratory Practice and Compliance ​

• We strictly follow the HKUST (GZ) Laboratory Safety Regulations in all procedures.
• Proper personal protective equipment, including lab coats, gloves, and safety goggles, is worn at all times in the laboratory.
• Chemical safety:
  • Preference is given to non-toxic or less toxic reagents, such as Safe Green and 4S Green Plus nucleic acid dyes, instead of traditional carcinogenic ethidium bromide.
  • For hazardous chemicals (e.g., isopropanol), members apply strict handling procedures, including designated fume hood usage, careful labeling, and waste segregation.

Culture of Safety ​

• Safety is not treated as a one-time requirement but as an ongoing practice. All team members are encouraged to proactively report hazards, reflect on safety practices, and support each other in maintaining a secure laboratory environment.

Biological Safety ​

Our project involves the use of commonly adopted laboratory model organisms, primarily Escherichia coli (strains DH5α, EPI400, top10, BL21, and DE3 ply) and Bacillus subtilis 168. Both species are classified as Biosafety Level 1 (BSL-1) organisms, widely recognized as non-pathogenic and safe for educational and research purposes.

Host and Organism Safety ​

• Escherichia coli: All strains used are standard laboratory derivatives that do not contain virulence factors and pose no pathogenic risks. They are commonly applied in cloning, protein expression, and educational research .
• Bacillus subtilis 168: Recognized as a GRAS (Generally Regarded As Safe) organism, frequently used in food and fermentation industries. Its genetic tractability makes it an ideal chassis for our synthetic pathways .
• Plant material: Lychee (Litchi chinensis Sonn.) was used as a natural substrate for application. No genetic modification of plants was performed.

Containment and Laboratory Practice ​

• All experiments are performed in BSL-1 facilities at HKUST (Guangzhou).
• Biological work is conducted under strict containment measures, including the use of open benches, biosafety cabinets, and chemical fume hoods when appropriate.
• Laboratory surfaces are disinfected before and after experiments, and all cultures, plates, and consumables are sterilized (autoclaved or chemically treated) before disposal.
• No live engineered organisms are released outside the laboratory. Our work fully complies with the iGEM White List and avoids any prohibited activities.

Safety Measures for Team Members ​

• All wet lab members received extensive training in biosafety protocols, including biological waste management, aseptic operation, and emergency response.
• Proper personal protective equipment (PPE), such as gloves, lab coats, and protective eyewear, is mandatory during all experiments.
• Team members not directly involved in wet lab work are officially restricted from laboratory access, as confirmed by our PI.

Containment Design ​

To minimize the risk of engineered Bacillus subtilis being released into the natural environment, we designed a phage-based biocontainment strategy.

Rationale ​

Natural phages that infect B. subtilis recognize the surface receptor protein YueB, which serves as the docking site for tail fiber proteins. However, using unmodified phages could also attack wild-type B. subtilis, an organism that is generally regarded as safe (GRAS) and beneficial in natural ecosystems. To avoid this, we engineered a synthetic recognition system.

Design ​

• Tail Fiber Engineering: The tail fiber proteins of the phage were artificially modified to recognize a redesigned variant of the YueB receptor .
• Host Engineering: Our engineered B. subtilis was also modified to express this synthetic YueB variant on its surface.
• As a result, only the engineered B. subtilis strain presents the correct “keyhole” for the engineered phage "key.”

Expected Outcomes ​

• Specificity: The engineered phage will only infect our engineered B. subtilis strains, leaving wild-type B. subtilis and other microorganisms unaffected.
• Environmental Safety: In the case of accidental release, the phage can be introduced to eliminate the engineered strains specifically.
• Reduced Ecological Risk: This complementary lock-and-key mechanism prevents broad infection and preserves the ecological functions of native B. subtilis.

Current Stage ​

This system is currently a design concept and has not been experimentally implemented. Nevertheless, it demonstrates our proactive approach to biosafety and our commitment to responsible innovation in synthetic biology.

Product and Risk Assessment ​

In addition to laboratory and biological safety, we evaluated the safety of our engineered products and strains to ensure they are suitable for potential applications in food preservation.

Product Safety ​

• Melatonin:
  • Naturally produced in many plants and already used as a dietary supplement .
  • Concentrations proposed in our project are within safe levels for human consumption. The U.S. Food and Drug Administration (FDA) recognizes melatonin as a dietary supplement ingredient, and the European Food Safety Authority (EFSA) has also evaluated its use in food .
  • No toxic by-products are generated in the biosynthetic pathway we designed.
• Wax coating:
  • Mimics natural fruit waxes, composed mainly of fatty acids and esters.
  • Similar waxes (e.g., carnauba wax) are widely used in the food industry as edible coatings and are recognized as safe.
  • Acts as a physical barrier to reduce water loss and microbial invasion without introducing harmful residues.

Strain Safety ​

• Chassis organisms: E. coli laboratory strains and B. subtilis 168 are both non-pathogenic and classified as BSL-1 organisms.
• No pathogenic genes or antibiotic resistance beyond standard markers were introduced.
• In the case of environmental release, our engineered strains do not pose significant ecological or health risks, and additional containment strategies (e.g., phage-based kill-switches) are planned.

Assessment and Responsibility ​

• We conducted literature reviews and consulted biosafety officers to confirm that both our final products and chassis organisms are safe under regulated use.
• This risk assessment ensures that our project does not only operate safely in the lab but also considers the safety of future consumers and the environment.

Legal & Regulatory Safety ​

Beyond laboratory and biological safety, our team also investigated the legal and regulatory framework surrounding the use of engineered microbes and their products in food preservation. With guidance from legal professionals, we conducted a preliminary compliance assessment.

GMO and Microbial Regulation ​

• According to the Administrative Measures for the Safety of Agricultural Genetically Modified Organisms in China, 2017, any genetically modified microorganism intended for industrial use must undergo strict safety evaluations before approval.
• Engineered B. subtilis strains cannot be directly released into the environment or applied to food products without official authorization. If commercialization is intended, safety assessments and approvals are mandatory.

Product Regulation ​

• Melatonin: In China, melatonin is currently regulated as a health supplement ingredient rather than a food additive. Its legal use is limited to dietary supplements, not direct application in fruit preservation.
• Wax coatings: Edible waxes such as carnauba wax (E903) and beeswax (E901) are approved under national food additive standards (GB2760-2014) . Our engineered wax must demonstrate equivalence to approved edible waxes in order to qualify for use.

Safety Standards and Testing ​

• If our engineered wax or melatonin coating is considered a food contact material (FCM), third-party toxicological testing would be required.
• National standards (e.g., GB/T45443-2025 for melatonin content measurement) and Codex Alimentarius guidelines would serve as reference frameworks.
• Any residues or metabolites exceeding legal thresholds could trigger regulatory restrictions or liability.

Future Considerations ​

• For commercialization, regulatory approval would be required both for the engineered microorganisms and their edible products.
• We recognize that regulatory landscapes differ across countries; while some regions (e.g., EU, USA) allow gene modified microbial products under strict conditions, others maintain stricter restrictions.
• By engaging legal professionals early, we ensure that our project anticipates possible compliance challenges and aligns with responsible innovation principles.

References ​