Overview
To test the functions and capabilities of our system TRAPS, we first developed it in Saccharomyces cerevisiae. All experiments were conducted under BSL-1 conditions provided by the lab of Prof. Simon Alberti (Alberti lab) in the BIOTEC Dresden. During all these steps we were in close contact with the safety officers and in compliance with the biosafety laws provided by iGEM and the German law. From the very beginning of our project, we integrated safety and security of our conducted experiments and our system into our planning to ensure that our work is responsible, reproducible, environmentally safe and in the guidelines provided by iGEM.
Laboratory Safety Practices
Training and Awareness
To ensure the safety of every member working in the laboratory, every member of our team involved in the laboratory completed comprehensive safety training before entering the laboratory. This involved the obligatory safety introduction provided by the Alberti research group, including the correct use of personal protective equipment, chemical handling, biological safety and emergency procedures. To ensure that everyone fully understood these concepts, we designed and implemented a biosafety quiz, which all members were required to pass before starting their lab work.
Risk management
To be best prepared for possible hazards regarding our project, we systematically analyzed risks in three main areas: chemical, biological and physical. Hazardous laboratory chemicals may pose toxic or irritant effects upon contact or inhalation. In this context, we also identified possible fire hazards, especially including the use of highly flammable substances. Beyond chemical risks, we consideres biosafety concerns such as the unintentional release of S. cerevisiae. Physical risks were also assessed, since laboratory instruments pose hazards under conditions of incorrect use. To minimize these risks, every team member involved in the laboratory work received the mentioned safety training prior to working in the laboratory. All experiments were conducted in restricted-access laboratories and under BSL-1 conditions. Standard laboratory practices were strictly followed, including the disinfection of workspaces, carefully monitoring of cultures and regular lab cleaning routines. All waste was collected, autoclaved and disposed according to the laboratory waste management plan. Hazardous chemicals were handled with special caution and all storage and waste management complied with institutional safety guidelines.
Organisms and Biological Parts
Saccharomyces cerevisiae
In our project we used Saccharomyces cerevisiae strain W303 (MATα) as our main laboratory organism. It belongs to the Risk Group 1 and is considered a safe model organism. S. cerevisiae was deliberately chosen over mammalian or immortalized cell lines because it provides the advantages of a eukaryotic system without the elevated biosafety concerns associated with higher organisms. All genetically modified strains used in our project were documented (“Formblatt Z”) according to the German legislation on genetically modified organisms (“Gentechnikgesetz/ GenTG”). To prevent the survival of the genetically modified S. cerevisiae outside of the laboratory, we implemented different auxotrophic strains. One risk we also had to think about was that under specific conditions S. cerevisiae tends to sporulation. Due to that risk, we developed a spore safety plan when working with S. cerevisiae.
Spore Safety Plan
Saccharomyces cerevisiae can form spores through a process called sporulation. This typically occurs under nutrient-limited conditions, especially when nitrogen is scarce and a non-fermentable carbon source (like acetate) is present. Only diploid cells can undergo meiosis and forming spores. Sporulation leads to the production of four resilient ascospores, which are enclosed within a protective sac called an ascus. This process helps yeasts survive harsh environmental conditions.
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Media and Growth Conditions
We exclusively used nutrient-rich media (YPD) that supports vegetative growth and does not promote sporulation. We avoided any media formulations or conditions known to induce sporulation, such as nitrogen starvation or the presence of non-fermentable carbon sources.
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Strain Selection
We used the laboratory strain of S. cerevisiae W303 (MATα) that is not specifically engineered for sporulation. This strain has been reported to have a significantly reduced tendency to sporulate under standard conditions (McNeill et al., 2025; Elrod et al., 2009).
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Expert Oversight and Training
Our project is carried out in close collaboration with experts who have worked with yeast systems for several years. They are trained to recognize early signs of sporulation and to respond appropriately to any potential events.
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Waste Handling
All cultures, waste, and materials that have come into contact with yeast were autoclaved before disposal. This ensured inactivation of any possible spores.
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Laboratory Containment
Work was conducted in BSL-1 designated areas with good microbiological practices, including the use of personal protective equipment (PPE) and proper cleaning protocols. Any spills were immediately disinfected with 70% ethanol.
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Monitoring and Documentation
Our team members are trained to recognize potential signs of sporulation. We regularly monitor culture conditions to ensure they remain within safe parameters. Any deviation from standard conditions must have been reported and addressed immediately.
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No Aerosol Generation
We avoided procedures that could lead to aerosol formation or environmental dispersal of cells, such as high-speed centrifugation without sealed rotors or agitation in open containers.
Through these combined measures, we ensured that sporulation was not only improbable but also safely contained in the unlikely case it were to occur.
Auxotrophic markers
To ensure biosafety also in moments of the unlikely event that genetically modified S. cerevisiae makes it way out of the lab, we relied on the S. cerevisiae W303 (MATα) strain, which is an auxotrophic strain. This strain lacks the ability to synthesize specific amino acids, making them dependent on the external supply of these amino acids only provided in laboratory conditions. By introducing our genetic constructs, some of these auxotrophic markeres were complemented, which allows growth on media lacking the specific amino acids. This enabled us to identify the colonies with integrated constructs. Our genetically modified S. cerevisiae W303 (MATα) strain was able to grow on media lacking uracil, leucine and tryptophan. While this reduces the number of auxotrophic markers, the used strain still cannot synthesize histidine and adenine and can therefore not survive on media lacking those amino acids. In addition, the strains remain laboratory organisms classified as Risk Group 1. Hence, their survival outside of controlled environments is still highly unlikely.
Escherichia coli
For plasmid amplification, we used Escherichia coli DH5-α, a non-pathogenic Risk Group 1 strain that is widely used in molecular biology. E. coli DH5-α was selected because it is one of the safest and most widely used E. coli strains for cloning and it is on the iGEM whitelist.
Future Risks and Responsible Use of TRAPS
TRAPS is a system designed to detect specific RNA molecules in real time within living cells. The system is intended to be only used as a new research tool for RNA detection under laboratory conditions and cannot be applied outside of the lab. In the future, we plan to integrate TRAPS into other cell types such as Danio rerio or mammalian cell cultures, where it could provide valuable insights into RNA expression under different conditions. For every new model organism, potential risks would need to be carefully evaluated. This includes evaluating the applicability of established safety measures and determining whether alternative containment strategies would be required to prevent accidental release into the environment. By consistently applying the same integrated biosafety approach as in our current work to any future adaptations of TRAPS, we aim to ensure that in future applications TRAPS can be used as a tool responsibly while maintaining strong biosafety standards.