Safety

Our experimental work took place in the laboratory of our Principal Investigator (PI), Prof. Vitor Pinheiro, situated at the Rega Institute for Medical Research on the Gasthuisberg Campus, just outside Leuven. This location, adjacent to the University Hospital UZ Leuven and the Leuven Fire Department, provides an added layer of assurance with swift emergency response capabilities. The Pinheiro lab, with its many years of experience supervising KU Leuven iGEM teams, operates under stringent ML-1 lab standards and adheres fully to the iGEM safety and security regulations.

All team members engaged in wet lab practices were required to undergo an extensive training program on Health, Safety, and Environment (HSE) matters. This training, overseen by a designated HSE liaison (Frederik) , equipped us with the necessary skills and legal authorization to work in the lab, as mandated by KU Leuven’s internal safety policies. This included comprehensive briefings on emergency protocols, such as handling hazardous spills, the safe disposal of biohazardous materials, and responding to fire emergencies. Additionally, team members received specific instruction on Good Microbiological Practices (GMP), with training provided by both experienced lab personnel and senior team members.

Particular attention was devoted to the responsible handling and disposal of reagents and materials, especially those containing organic solvents and acids, as outlined in our colorimetric protocols. Although our work was under the supervision of trained lab personnel, we implemented additional proactive safety measures to mitigate risks during daily lab operations. We developed detailed Safe Machine Usage (SMU) protocols (see link) and instituted a rotational daily auditing system to continuously monitor critical laboratory “hotpots” (See hotspots list) ensuring compliance with safety standards.

For our experimental work, we utilized Escherichia coli strain DH10β, a derivative of the K12 strain. Classified as a BSL-1 organism, DH10β presents minimal risk to human health and the environment. Due to the deletion of the leuLABCD gene cluster, it is leucine-auxotrophic, and its relA1 and spoT1 alleles further reduce its growth rate and sensitivity to nutrient depletion. These genetic traits contribute to its poor survival in natural environments, ensuring it does not pose a threat to other microorganisms or plants. Research consistently demonstrates that K12-derived strains, including DH10β, exhibit minimal environmental impact.

Following the successful cloning of our metal-binding library, our focus shifted towards the screening and measurement of the metal removal efficiencies of the proteins we developed. We took great care to design robust metal measurement protocols (refer to colorimetric protocols), with an emphasis on safety, precision, and reproducibility. All handling of hazardous compounds was conducted within a fume hood and closely supervised by specialist chemists, ensuring that all safety procedures were followed meticulously.

At iGEM KU Leuven, we hold ourselves to the highest safety standards, fostering a culture of responsibility and diligence. By integrating rigorous training, continuous monitoring, and proactive safety protocols, we have created a lab environment that prioritizes both scientific integrity and the well-being of our team.

Neominex ™ solution has been designed to treat confined industrial waters, but after discussing with iGEM judges and our industry network (see human resources), we decided to design a kill-switch in case a leak out happens. The kill-switch is a logic gate NOR system based on two separate inputs: temperature and quorum sensing (figure 1X to 3X) (3, 4, 5).

Why separating the kill switch in these two inputs? This kill switch was designed to quickly control biocontamination of large water bodies. If an escape of the genetically engineered microbe (GEM) culture occurs, the CFU/mL value will shrink temporally until the culture starts dividing again (if possible). Also, the temperature of a large mass of water in nature is frequently lower than 37ºC, as these liquid temperatures are only found in isothermal animals and in volcanic active zones. By capitalizing on these two ideas, the switch aims to trigger only if both conditions (low cell density and lower than 37ºC temperature) are held at the same time.

Although in the future it would be interesting to move away from E. coli and use other bacteria with enhanced metal toxicity and lower temperatures of growth, now this kill switch would allow for the treatment of industrial wastewater using moderate temperatures (37ºC), reducing the risk of relishing our GEM to the environment.