Introduction

Biosafety is a fundamental aspect of synthetic biology projects, as it ensures the safety of researchers, the environment, and society from the risks associated with handling living organisms and laboratory reagents. In the context of the iGEM competition, the implementation of biosafety measures ensures that research complies with the highest international standards of scientific safety and social responsibility.

This report describes the biosafety strategies adopted by the iGEMUAM team in the framework of its project, whose objective is the development of a system for the production of antibody fragments (scFv) in the yeast Pichia pastoris as the basis for a potential antivenom. This approach takes advantage of a non-pathogenic eukaryotic organism widely used in biotechnology, which helps to reduce the risks associated with the use of more dangerous biological agents.

Throughout the document, the organisms and materials used, the risks identified, the prevention and mitigation measures implemented, and compliance with national and international biosafety regulations are presented. In doing so, the team reaffirms its commitment to conducting safe, responsible scientific work that has a positive impact on society.

Project Description and Organisms Used

The iGEMUAM team's project seeks to develop an innovative strategy for the production of single-chain variable fragments (scFv) with potential application in the generation of antivenoms. Traditionally, antivenoms are obtained by immunizing large mammals (such as horses) and purifying immunoglobulins, a process that involves high costs, intensive use of animals, and variability in product quality. Our approach proposes the use of synthetic biology techniques and recombinant organisms as a more sustainable, scalable, and ethical alternative.

We seek to achieve this with the selected host organism Pichia pastoris yeast (also known as Komagataella phaffii), a eukaryotic expression system widely used in industrial biotechnology for its ability to efficiently secrete recombinant proteins, perform post-translational modifications, and grow under simple and low-cost conditions. It should be noted that P. pastoris is considered a non-pathogenic and laboratory-safe organism, classified within biosafety level 2 (BSL-2).

For the initial stages of cloning and assembly of genetic constructs, Escherichia coli K-12 is used, a non-pathogenic laboratory strain, standard in molecular biology and recognized as safe. The DNA fragments used include coding sequences for scFv, promoters, secretion signal regions, and an expression vector derived from pPICZαA, which is optimized for Pichia pastoris.

Overall, the experimental design allows for safe work, as both the organisms and the molecular tools selected have been extensively tested and validated in low-risk laboratory settings.

Risk Identification (summary)

Detailed category pages are available under Safety: Biological, Chemical, and Physical.

Biological risks: E. coli (K-12, non-pathogenic; used for cloning), P. pastoris (non-pathogenic; industrially used). No virulence/toxin genes included. Identified risk: accidental contamination or improper handling.
Chemical risks: Reagents such as SDS and selection antibiotics (Zeocin). Identified risk: skin exposure, inhalation, spills.
Physical risks: Centrifuges (imbalances), electroporator (electric shocks), UV sources (skin/eye damage). Identified risk: accidents from improper use or missing PPE.
Environmental risks: Possible accidental release of GMOs; low risk but requires proper containment and disposal.

Biosafety Measures Implemented

Laboratory biosafety level: Activities in BSL-1 labs suitable for E. coli (DH5α/XL1-Blue) and P. pastoris. Restricted access to trained personnel.
Personal protective equipment (PPE): Lab coats, nitrile gloves, safety glasses; masks as needed.
Waste management: Biological waste inactivated by autoclaving prior to disposal.
Training and supervision: Training in biosafety, biological material handling, and waste disposal; incident reporting.
Safe working procedures: Culture handling in designated areas; use of biosafety cabinets/ventilated areas for aerosols/volatiles; proper centrifuge balancing and supervised electroporation.
Signage and control: Visible hazard signage; restricted access to biological areas.

Regulations and Compliance

See detailed items in the Legislation pages (National, International, iGEM).

National (Mexico): GMO Biosafety Law; NOM-087-ECOL-SSA1-2002 (RPBI); NOM-005-STPS-1998 (lab safety); NOM-012-SSA3-2012 (health research); NOM-164-SEMARNAT/SAGARPA-2013 (GMO releases); NOM-056-SSA1-1993 (PPE).
International: WHO/NIH biosafety principles; HACCP; SPS Agreement; Cartagena Protocol.
iGEM: Safe Project Design; Safe Lab Work; social and environmental responsibility.

Risk Assessment and Mitigation

Biological: Low probability/minor consequences. Mitigation: designated culture areas, autoclave disposal, ongoing training.
Chemical: Medium probability/moderate consequences. Mitigation: specialized PPE (chemical-resistant gloves, masks, face shields), ventilated cabinets for volatile solvents, segregated labeled disposal.
Physical: Low–medium probability/mild–severe consequences. Mitigation: supervised use of high-risk equipment, training on centrifuge balancing/electroporation, mandatory eye protection near UV.
Environmental: Low risk due to non-pathogenic species. Mitigation: primary containment (safe pipetting, biosafety cabinets), secondary containment (restricted areas, signage), autoclave all cultures.

Emergency Plan

Biological spills

Cover the affected area with absorbent towels.
Apply disinfectant (70% ethanol or sodium hypochlorite) and leave for ≥ 20 minutes.
Remove material with gloves; place in RPBI containers for autoclaving.
Notify the biosafety officer and record the incident.

Chemical spills

Evacuate the immediate area if volatile/corrosive (e.g., phenol, chloroform).
Use a chemical spill kit (special absorbents).
Dispose of contaminated material in labeled hazardous waste containers.
On skin/eye contact, rinse with water and seek medical attention.

Physical accidents

Electrocution: cut power before assisting.
Centrifuge malfunction: stop equipment; do not open until complete stop.
UV exposure: turn off source, remove affected person, seek medical evaluation.

First aid and contacts

Maintain a basic first aid kit and train the team in first aid.
Report injuries immediately to the biosafety supervisor.
Emergency contacts: university or local medical services.

Impact and Social Responsibility

Scientific/technological: Yeast expression systems offer sustainable, scalable alternatives to animal-derived antivenoms; contributions to synthetic biology tools for public health.
Social: Ethical approach reducing large animal use (3Rs), potential for improved antivenom accessibility in high-need regions.
Environmental responsibility: Use of non-pathogenic organisms and strict disposal protocols minimize environmental risks.
Ethical commitment: Work under biosafety/bioethics/current regulations; transparent communication to the scientific community and public.

Conclusions

This biosafety report reflects the iGEMUAM team's commitment to safe, responsible scientific research aligned with national and international standards. The potential biological, chemical, physical, and environmental risks associated with the project were identified, and mitigation measures and emergency plans were established to protect the team, the community, and the environment.

The use of non-pathogenic organisms such as Escherichia coli and Pichia pastoris, together with appropriate containment and disposal protocols, ensures that experimental activities are carried out under conditions of minimal risk. Ongoing team training, responsible waste management, and compliance with current regulations further strengthen project safety.