Safe Lab Work

Comprehensive Safety Measures in Mhetyguá Project

In the realm of scientific research, particularly when delving into the transformative field of genetic engineering, safety is paramount. As we embark on our ambitious journey to address the pressing issue of microplastic pollution using genetically engineered microalgae, we recognize the profound responsibility that accompanies our endeavors. It's not just about pioneering innovative solutions; it's about ensuring that every step we take is grounded in rigorous safety protocols, ensuring the well-being of all stakeholders involved. The following details provide a comprehensive overview of the meticulous safety measures we've integrated into our project, reflecting our unwavering commitment to responsible and ethical research.

1. Rigorous Adherence to Established Guidelines:

Our project is anchored in strict compliance with established biosafety guidelines. We've ensured that our chosen organism, Chlamydomonas reinhardtii, is safe for genetic engineering. This microalgae has been designated as Generally Recognized As Safe (GRAS) by the United States Food and Drug Administration (FDA). This designation is a testament to its safety, especially when used under controlled conditions.

2. Safe Organism Selection:

Chlamydomonas reinhardtii is a single-celled microalgae that poses minimal biosecurity risks. It does not cause diseases in humans or animals. Its non-pathogenic nature is a cornerstone of our project's safety profile. Furthermore, its limited ability to survive and reproduce outside a controlled laboratory environment ensures that there's minimal risk of unintended release or proliferation in natural ecosystems.

3. Specialized Training and Expertise:

All team members undergo rigorous training, ensuring they are well-equipped with the necessary knowledge and skills to handle genetically modified organisms safely. This training encompasses the correct use of personal protective equipment (PPE), waste management protocols, and emergency response procedures. Our team's expertise is further bolstered by the guidance of seasoned mentors and professors, some of whom serve on esteemed biosafety commissions.

4. Advanced Laboratory Infrastructure:

Our state-of-the-art laboratories are equipped with essential tools and equipment, including biosafety hoods, centrifuges, and incubators. These facilities are specifically designed to mitigate risks, ensuring that all experimental procedures are conducted safely and efficiently.

5. Waste Management Protocols:

We have established stringent protocols for the collection, storage, and disposal of waste. All materials that come into contact with genetically modified organisms are sterilized to prevent any potential environmental release. This meticulous approach to waste management underscores our commitment to environmental stewardship.

6. Enzyme Safety and Specificity:

The enzymes we employ, specifically designed to degrade plastics like PET, have undergone extensive research and testing. Their efficiency and specificity in breaking down PET have been well-documented, ensuring that they don't inadvertently affect other substances or ecosystems.

7. Continuous Monitoring and Documentation:

Our project emphasizes transparency and traceability. We maintain detailed records of all experimental procedures, ensuring that any anomalies or deviations can be promptly identified and addressed. Regular monitoring of cultures, rigorous disinfection protocols, and consistent documentation practices ensure the highest standards of safety and accountability.

8. Dual-Use and Risk Management:

We recognize the potential dual-use implications of any scientific endeavor. To address this, our project is conducted under the watchful eyes of experienced mentors, advisors, and professors. Their expertise ensures that our research remains focused on its intended purpose, minimizing any potential for misuse or unintended consequences.

9. Commitment to Public Safety and Environmental Protection:

Our project's ultimate goal is to address the pressing issue of microplastic pollution in aquatic environments. Every step, from the selection of organisms to the design of genetic circuits, is taken with a keen awareness of its potential impact on public health and the environment. Our commitment to safety is unwavering, and we continually strive to ensure that our research benefits society without introducing new risks.

Kill-switch Device

Although Chlamydomonas reinhardtii is non-pathogenic, it has been genetically modified to function as an effective microorganism for bioremediation. To prevent the escape and survival of these modified algae into the environment, we have developed a safety system that utilizes the cyanobacterial non-specific DNA/RNA nuclease NucA and its inhibitor NuiA from Anabaena.

Upon exposure to NaCl, NucA is activated and breaks down all DNA/RNA in the microalgae, preventing any unintended release of the genetically modified organisms. In the closed system, the presence of the NuiA inhibitor keeps the nuclease inactive under normal conditions.

To ensure conditional expression and cell survival in the photobioreactor or open pond, while also preventing survival upon accidental release into the environment, we rewired the nuclease/inhibitor pair. In this system, the nuclease gene was placed under GPDH3 an inducible promoter, allowing for induction upon exposure to environmental inducers.

To safeguard against possible leaky nuclease production in the bioreactor in the absence of an inducer, the nuclease inhibitor gene was fused to a weak constitutive promoter. This additional layer of safety ensures that the nuclease remains inactive under normal conditions in the closed system.

References

• Beltran-Aguilar, A. G., Peraza-Echeverria, S., López-Ochoa, L. A., Borges-Argáez, I. C., & Herrera-Valencia, V. A. (2019). A novel salt-inducible CrGPDH3 promoter of the microalga Chlamydomonas reinhardtii for transgene overexpression. Applied microbiology and biotechnology, 103(8), 3487-3499.
• Čelešnik, H., Tanšek, A., Tahirović, A., Vižintin, A., Mustar, J., Vidmar, V., & Dolinar, M. (2016). Biosafety of biotechnologically important microalgae: intrinsic suicide switch implementation in cyanobacterium Synechocystis sp. PCC 6803. Biology Open, 5(4), 519-528.
• ANAYELI GUADALUPE BELTRAN-AGUILAR et al. A novel salt-inducible CrGPDH3 promoter of the microalga Chlamydomonas reinhardtii for transgene overexpression. Applied Microbiology and Biotechnology, v. 103, n. 8, p. 3487–3499, 21 mar. 2019.