We prioritize safety and transparency in all aspects of our work. Our dual modular oral immunization platform against avian influenza A H5N1 uses Lactococcus lactis (GRAS) as the chassis and combines two innovative strategies.
Introduction
Safety First
Identifying Possible Risks
Risk Assessment
After careful evaluation, we identified the potential risks associated with our project:
1. Ciclobodies
Synthetic bivalent nanobodies stabilized by inteins for direct viral neutralization.
2. Surface Display
Display of influenza antigens to trigger mucosal and durable adaptive immunity.
Modular expression units, driven by optimized promoters and regulators, allow inducible expression and rapid replacement of immunogenic parts as new variants emerge.
Potential risks include handling of viral parts (HA/NA domains, classified as RG2 but non-infectious) and the use of engineered microorganisms at BSL-1/2. Hazards could arise from chemical exposure (NaOH, CTAB, SDS, alcohols), accidental release of genetically modified microorganisms, or general laboratory accidents such as burns and cuts.
The organisms involved are Escherichia coli DH5α (for cloning), BL21(DE3)/C41(DE3) (optional for protein expression tests), and Lactococcus lactis subsp. lactis ATCC 19435™ (final chassis). Viral sequences (neuraminidase stalk domain, HA2 long α-helix with M2e repeats) will be codon-optimized, commercially synthesized by an IGSC member, and expressed in L. lactis. No viral replication or packaging elements are used.
How We Eliminate These Risks
Risk Control Measures
All planned experiments will be carried out under BSL-1 or BSL-2 conditions depending on the material used. Standard assays include cloning in E. coli, transformation and expression in L. lactis, SDS-PAGE, Western blotting, immunofluorescence, ELISA for antigen display, and in vitro growth and stability characterization.
Risk controls include the use of GRAS organisms, good microbiological practices, biosafety training, PPE, autoclaving of biological waste, inventory tracking, and incident reporting. Future field applications such as poultry vaccination through water or feed would only take place after iGEM, under appropriate institutional and national approvals.
AI is used only as an auxiliary tool for design and prediction. Human oversight, literature verification, and experimental validation ensure that AI does not make final decisions.
External experts from Ecuador's Ministry of Environment (MAATE) and Agrocalidad have been consulted, and their guidance has shaped our biosafety approach.
Our Delivery Platform
Safe Research Design
Our project focuses on the delivery of immunogenic parts. The system is designed for safe research use and does not transfer harmful elements such as pathogenic proteins or viral factors. Conjugation-based methods remain at low risk as long as experiments do not exceed BSL-2 thresholds.
The chassis and plasmids used are classified as BSL-1, and additional safeguards such as nutrient-dependence are considered to prevent persistence in the environment. Any potential poultry applications will explicitly require approval from MAATE and Agrocalidad before implementation.
Our Lab
Laboratory Safety & Facilities
Safety in Our Lab
All work is carried out in laboratories equipped with open benches, biosafety cabinets, and chemical fume hoods. Access is restricted and materials are logged for full traceability. Biosafety training covered containment, disinfection, sterilization, emergency response, dual-use awareness, and incident reporting.
Waste Disposal
Biological waste is autoclaved before removal, while chemical waste is segregated and managed by certified handlers. All storage and labeling follow institutional guidelines.
Details
Team members are required to wear lab coats, gloves, and protective eyewear at all times. Work is conducted under the two-person rule when necessary, and lone-worker policies are in place. Emergency protocols and data security procedures are established to ensure safe laboratory operations.
Our Instruments and Machines
The project uses biological safety cabinets for GMM work, autoclaves for sterilization, and chemical fume hoods for hazardous reagents. Each instrument is operated according to institutional manuals and training provided by laboratory experts. To further improve lab safety, new infrastructure such as a gel cutting station was implemented to replace traditional UV-based cutting methods, reducing exposure risks.
Chemicals
Safe Handling Procedures
All chemicals are registered in the institutional safety system and managed in compliance with national and European standards. Safety data sheets are available for all reagents, and storage is controlled.
Hazardous substances such as ethanol, isopropanol, Tris-HCl, NaOH, CTAB, SDS, and enzymes like RNase A are handled with strict precautions. For nucleic acid staining, SYBR Safe is used instead of ethidium bromide to minimize risks. Access to restricted chemicals is limited to authorized team members and kept in separate compartments.
Compliance and Forms
Regulatory Framework
The project complies with iGEM and institutional biosafety regulations:
No RG3/4 organisms, no human testing, and no environmental release are performed during iGEM.
Use of viral parts (HA/NA) requires an official Check-In, which has been requested as per iGEM policy.
Any future poultry applications will require additional Animal Use and Release approvals from national authorities.
DNA synthesis is ordered from an IGSC member company to ensure compliance.