Biology is a rich scientific discipline full of possibilities. However, it is not without risk. To carry out a good scientific project, it is important to assess hazards and protect against accidents that could occur.
Below you will find all the measures we have taken to ensure that our iGEM adventure proceeds safely. Be innovative and entrepreneurial — but be safe, and you will be so for longer.
Biological risks and mitigation measures
Non-pathogenic chassis: Our project uses Streptococcus thermophilus as the chassis organism. This choice was motivated by biosafety considerations: S. thermophilus is a non-pathogenic lactic acid bacterium recognized for its use in food (yogurts, cheeses) and holds GRAS status (Generally Recognized As Safe). This species belongs to risk group 1, which means it does not pose a danger to healthy humans. Using such an organism immediately reduces risks of pathogenicity or toxicity: S. thermophilus has no known infectious capacity and its environmental impact is limited, especially since it is adapted to specific environments (milk) and survives poorly outside controlled conditions.
Microorganisms used: In addition to S. thermophilus, we also used the Escherichia coli TOP10 strain for certain technical aspects (for example, the production and purification of fluorescent proteins used as controls). E. coli TOP10 is also a risk group 1, non-pathogenic organism, commonly used in laboratories. Using biosafety level 1 strains for all our microorganisms ensures that no pathogen or potentially infectious agent was handled in the project.
Genetic elements introduced: The genetic constructs we introduced into S. thermophilus include genes encoding fluorescent proteins (mScarlet, mTurquoise), nanobodies (single-domain monoclonal antibodies), and Alpha-Rep binding proteins, as well as a fucanase enzyme. None of these elements is toxic or increases the strain’s virulence. We deliberately refrained from using genes likely to produce toxins, allergens, or other dangerous molecules. Thus, the genetic parts selected for our project minimize risks to humans, animals, and the environment, in line with iGEM recommendations.
Antibiotic resistances: To select modified bacteria in the laboratory, we used antibiotic resistance genes (notably low-concentration chloramphenicol resistance, 5 µg/mL, suitable for S. thermophilus). We are aware that introducing an antibiotic resistance gene is a potential biological risk, particularly if this gene were to be transferred to other environmental microorganisms (horizontal gene transfer). To manage this risk, we took several precautions:
- Strict confinement of modified strains: all manipulations take place in a biosafety level 1 laboratory, and no genetically modified strain leaves the lab.
- Rigorous management of biological waste: all culture media, bacteria, and contaminated materials are disinfected and autoclaved after use, in order to kill bacteria and degrade DNA, preventing any spread of resistance genes into the environment.
- Curable plasmid and linear DNA: wherever possible, we favored approaches that do not leave permanent resistance: for example, since S. thermophilus is naturally competent, we used natural transformation to integrate linear DNA fragments into the genome (from PCR with homology arms), which avoids prolonged use of resistance-bearing plasmids. In cases requiring a plasmid, we used a transient episomal vector that can be eliminated after use (such as the pGhost9 plasmid, known to be thermosensitive and curable). This strategy means antibiotic resistance is only required temporarily for selection: once the construct is integrated and confirmed, the plasmid can be lost, ideally leaving no active resistance gene in the final strain.
These measures greatly reduce the risk that an antibiotic resistance used in the lab could spread or persist outside the controlled environment.
Chemical risks and handling of hazardous substances
Chemicals used: The molecular biology and microbiology activities in our project involve common laboratory chemical reagents. Among them: culture products (growth media such as M17 agar supplemented with specific sugars), PCR reagents (enzymes, buffers, etc.), DNA stains for gel electrophoresis, and other products such as ethanol, PEG or sodium acetate solutions (for DNA purifications), as well as protein-purification reagents (for example imidazole solutions and Ni-NTA chromatography resins when purifying a His-tagged GFP). Most of these substances do not present high danger if handled correctly, but we nevertheless took great care to follow chemical safety instructions for each product.
Chemical safety measures: In accordance with good laboratory practices, all team members wore appropriate personal protective equipment (PPE) during manipulations: long-sleeved lab coat, nitrile gloves, and safety glasses for at-risk procedures (for example when using irritant substances or working near a UV source for gels). In particular, we ensured:
- Avoiding or substituting the most hazardous agents: for DNA analysis on agarose gels, we used a non-mutagenic stain (SYBR Safe-type) rather than traditional ethidium bromide, to reduce toxic and mutagenic risks for people and the environment. Likewise, flammable solvents (ethanol, isopropanol) were handled away from flames or heat sources, and in small quantities.
- Careful handling and secure storage: chemical reagents (notably culture-medium powders, which can be irritant, or acrylamide used for SDS-PAGE gels, which is toxic when unpolymerized) were handled under a fume hood when necessary and stored in appropriate containers (ventilated cabinets or secured flammables storage). Each bottle was clearly labeled with safety pictograms.
- Proper disposal of chemical waste: waste containing hazardous products (for example agarose gels containing a DNA stain, or acrylamide solutions) was collected separately and disposed of through our institution’s chemical waste stream to prevent any environmental contamination.
By following these measures, no chemical incidents occurred during the project. The attention paid to material choices (substituting harmful substances when possible) reflects our commitment to complying with iGEM recommendations on using safer materials in experiments. Every team member received training on chemical risks at the start of the project, ensuring informed and safe handling of all reagents.
Physical risks and laboratory safety
The biology laboratory in which we worked presents some physical risks inherent to the use of equipment and the handling of sharp or hot objects. We identified the following and applied the corresponding instructions:
Electrical equipment and machines: We used devices such as centrifuges, shakers, a thermocycler (PCR), an electrophoresis system and its UV transilluminator, incubators (including a shaking incubator at 37 °C for bacterial cultures), as well as an autoclave for sterilization. Each piece of equipment was used by members trained in its use, conscientiously and in accordance with the manufacturer's recommendations. For example, for the centrifuge, we always ensured correct balancing of samples and tightly closed the lid before starting, to avoid imbalance or accidental opening during rotation. Only authorized personnel who were familiar with emergency protocols handled the autoclave (wearing heat-resistant gloves, releasing pressure before opening, etc.), given the risk of burns and overpressure associated with this device. Likewise, when using the UV transilluminator to visualize DNA gels, UV safety glasses and a face shield were worn to prevent any exposure of eyes or skin to harmful UV rays. These precautions are intended to eliminate risks of accidents or injury when using laboratory equipment.
Sharps and glassware: Protocols involved the use of glass Pasteur pipettes, scalpel blades (for example to excise agarose gel bands), and syringes or needles for certain sampling steps. All these sharp/cutting items were handled carefully. We set up a sharps container to safely dispose of items after use, thus avoiding injuries and ensuring that no hazardous waste ends up in regular bins. Glassware (beakers, Erlenmeyer flasks) was inspected before use to ensure the absence of cracks, and we wore gloves for hot handling (e.g., removing sterilized material from the autoclave) to avoid cuts or burns.
Ergonomics and other risks: We kept the workspace tidy to avoid falls or collisions. Electrical cables were kept clear of walkways. No experiment involved extreme physical risk (no ionizing radiation, no heavy industrial machinery, etc.), but the team followed general rules: never working alone late at night without supervision, knowing the location of the first-aid kit and the emergency shower/eyewash, etc.
Security risks and protection against misuse
In the iGEM context, “security” refers to the risks that a project or technology could be misused for malicious or irresponsible purposes. We assessed our project from this angle and found that the potential for misuse is extremely low.
Our system uses S. thermophilus, a harmless bacterium, modified to express proteins of interest (fluorescent proteins, nanobodies, etc.) useful in research or biotechnology applications. None of the functions we confer on this strain gives it a dangerous advantage or harmful capability: for example, we do not introduce resistance to a clinically critical antibiotic (chloramphenicol is no longer commonly used clinically and is used here only at low dose for lab selection), and we do not insert virulence genes or genes producing toxic compounds. It would therefore be difficult to imagine a scenario in which a malicious actor would use our modified strain as a biological weapon or to cause harm.
Nevertheless, we took additional precautions to ensure project security: all modified strains and biological materials are kept in a secure laboratory under the supervision of our supervisors. Access to the laboratory is restricted to authorized, trained members. No biological samples were sent to third parties outside controlled channels (the only shipments of biological material concerned plasmids/DNA for sequencing to approved providers, in accordance with DNA transport regulations and without sending live bacteria).
We also reflected on the ethical and security implications of producing HMOs for commercial purposes. Indeed, aiming to improve food products may seem risk-free; however, narrowing the gap between formula milk and breast milk could have consequences for consumer habits. During our various interviews with infant-health experts, we grasped the major importance that breast milk can play in babies’ development. This is why, in each of our public communications, we specified that it is essential to prioritize nutrition with breast milk and that formula should be used as a last resort. This messaging must remain central in the event that we consider marketing a future product.
Built-in safety features in project design
Several design features of our project were conceived from the outset to maximize safety:
- GRAS chassis choice: As mentioned, using S. thermophilus (GRAS, non-pathogenic) was a deliberate choice to avoid risks associated with opportunistic or dangerous organisms. This choice aligns with iGEM recommendations to work with non-pathogenic chassis and is justified by the fact that S. thermophilus allowed us to achieve our scientific goals while minimizing biosafety concerns.
- Use of natural competence: S. thermophilus is naturally competent, meaning it can take up DNA from its environment and integrate it into its genome. We leveraged this ability by introducing our genetic constructs as linear DNA (from overlap-PCR with homologous arms) rather than systematically via circular plasmids. This approach has a safety advantage: free linear DNA is quickly degraded outside the cell and cannot replicate autonomously, which limits the risk of dispersing modified genetic material outside the laboratory. Moreover, it allowed us to avoid the use of multi-copy plasmid vectors which, if they escaped, could persist longer in the environment. In short, natural transformation made our modifications more contained and controlled.
- Programmable plasmid elimination: When a plasmid was necessary (for intermediate steps or transient expression), we chose the pGhost9 plasmid. This vector is thermosensitive and can be eliminated from the strain by changing culture conditions (shift to a non-permissive temperature). Thus, it is a “curable” plasmid, designed not to remain permanently in cells. This feature is an important biosafety element: after accomplishing its function (e.g., enabling selection of a genomic integration via a resistance marker), the plasmid can be cured from the strain, simultaneously removing the antibiotic resistance gene it carries. In our experimental design, this opens the possibility of ultimately obtaining a modified strain without residual antibiotic resistance genes, which is desirable if one envisages any application outside the strict laboratory setting.
- Modular genetic parts and expression control: Our genetic constructs include well-characterized promoters and regulators, allowing control over the expression of inserted genes. For example, potentially foreign proteins (Alpha-Rep, nanobody) can be expressed conditionally or at moderate levels so as not to disturb the bacterium’s physiology beyond what is necessary. This is part of our approach to minimizing stress on the strain, to avoid the emergence of unforeseen mutations or deleterious effects that could indirectly pose safety issues.
- Absence of hazardous “gain-of-function”: We verified that the modifications made do not grant new unpredictable capabilities to the organism. For example, deletion of the htrA gene (encoding an HtrA protease) was undertaken to improve the stability of expressed heterologous proteins, not to change the bacterium’s viability or robustness. This modification does not confer a dangerous gain of function (it is rather a loss of function that may slightly weaken the bacterium under stress conditions). Similarly, expression of fucanase aims to degrade a specific polysaccharide in the medium, which is not in itself an advantage in a natural environment for the bacterium and does not make it more invasive or resistant.
Verification and compliance with safety standards
In addition to prevention and safe design, our team paid close attention to verifying the results obtained and complying with safety standards throughout the project:
PCR and sequencing checks: To ensure that the genetic modifications made were exactly as planned and that no undesirable element had been introduced, we systematically verified our constructs by PCR and sequencing. For example, after each step of integrating a DNA fragment into S. thermophilus, we performed colony PCR or PCR on extracted DNA to confirm the presence of the fragment at the correct locations, then had the products sequenced to validate the absence of mutations or unexpected rearrangements. This verification step ensures not only the scientific integrity of the project but also safety: by knowing precisely the genetic sequence of the modified strains, we ensure that no potentially problematic element (e.g., an unintended piece of vector, or a mutation conferring an unforeseen phenotype) is present. If anomalies had been detected, we could have decided not to continue with those strains. Fortunately, sequencing confirmed that our constructs matched the expected design.
Training and supervision: All team members completed initial training in laboratory safety rules at the start of the project. This training, provided by our supervisors and our institution’s health and safety officer, covered basic practices (sterilization, disinfection, PPE, emergency procedures) as well as instructions specific to the use of S. thermophilus and E. coli. In addition, our experiments were supervised by experienced researchers who regularly ensured compliance with safety protocols. One or two team members were identified as “safety” contacts to remind everyone of best practices on a daily basis.
Regulatory and iGEM compliance: We ensured compliance with all iGEM safety requirements. Since our project involves only risk group 1 organisms, no special authorization was required beyond the standard declaration of the strains used. We completed the iGEM safety questionnaire detailing our use of S. thermophilus, notably to inform the Safety & Security Committee of our choice of a chassis less common than E. coli but equally safe. This choice was validated, with no additional check-in required for a GRAS, risk group 1 species. Furthermore, we strictly followed iGEM safety policies by avoiding prohibited materials (no synthetic DNA fragments beyond authorized lengths without validation, no use of whole organisms in animals, etc.) and by meeting deadlines — for example, the final safety form was submitted before the deadline, in accordance with competition rules.
Documentation and transparency: We have documented all our safety procedures on this page and in our lab notebook in a transparent manner. This allows both iGEM judges and the public to verify our compliance and understand how we integrated the safety dimension into the project. In case of questions or an audit, all information (inactivation protocols, substance safety data sheets, proof of training, etc.) is available or archived on our premises.