Safety and Security
Safety
Safety is the core principle of our experimental project and the foundation of its success. We are deeply aware that in the field of synthetic biology, safety is not just a project requirement but also our responsibility to society and the environment. Therefore, from chassis organism selection and project design to experimental operations, we have applied rigorous safety strategies at every step to ensure that our "Yeast Medics" project benefits patients while minimizing potential risks to users and the environment.
Before the project commenced, during the design phase, a comprehensive assessment of potential biological risks, reagent toxicity, chemical risks, etc., was conducted to ensure all materials and procedures meet safety standards. Not only did the wet lab team members receive thorough safety training, but hardware group members who brought robotic arms into the lab for disinfection and testing also underwent complete safety training, passed the laboratory access permission exam, and possess laboratory safety qualifications, ensuring their operational safety competence in the lab.
Through these measures, we can ensure the safety of the experimental process and have maintained a safety consciousness throughout subsequent Human Practices activities. Our goal is to conduct high-quality scientific research and to set an example for safety management in future synthetic biology projects.
1. Chassis Organism Safety
The chassis organism we selected is the Yarrowia lipolytica Po1h strain. This strain has extremely high biosafety. As a Generally Recognized As Safe (GRAS) strain: Yarrowia lipolytica is a non-pathogenic, non-conventional yeast that has received GRAS certification from the US FDA and is widely used in the food and pharmaceutical industries, is considered to pose no harm to humans or the environment(Park & Ledesma-Amaro, 2023). Although, as an industrially promising strain, this yeast can tolerate the high-sugar and oxidative stress conditions of a wound environment, its auxotrophic nature fundamentally prevents its survival and reproduction in the external environment(Arnesen & Borodina, 2022).
2. Project Design Safety
We incorporated the philosophy of "Safety by Design" at the project design stage, ensuring system controllability and safety through multiple strategies:
2.1 Genetic Operation Safety:
Plasmid Choosing:
The plasmid system (pINA1317) we employed is an integrating plasmid specifically designed for Yarrowia lipolytica Po1h, which exhibits high genetic stability and does not require antibiotic selection pressure during cultivation (Yue et al., 2008). Although this plasmid carries a kanamycin resistance marker gene, we excised the fragment containing the kanamycin resistance gene using Not I restriction enzyme prior to yeast integration. This step prevents its incorporation into the yeast genome, thereby eliminating any potential risk of contributing to antibiotic resistance or environmental contamination. All heterologous genes (the antimicrobial peptide Pexiganan, IL-4, and VEGF) are derived from human or other well-characterized safe sources, and their functions are thoroughly documented in the literature, precluding unpredictable toxicity or side effects.
Img.1 Plasmid: pINA1317
Physical Encapsulation and Leakage Prevention:
We developed a thermosensitive hydrogel, L-HBC, based on hydroxybutyl chitosan (HBC), as a delivery vehicle for the engineered yeast(Sun et al., 2020). Our experimental validation indicates that this hydrogel effectively encapsulates yeast cells displaying the antimicrobial peptide on their surface, with a very low leakage rate, effectively preventing the engineered yeast from entering the patient's body or the external environment.
Img.2: the leakage experiment's result of 24h
2.2 Therapeutic System Safety:
We chose the antimicrobial peptide Pexiganan instead of traditional antibiotics, mechanistically avoiding the risk of promoting antibiotic resistance. The expression of cytokines IL-4 and VEGF is strictly regulated by sugar concentration and infrared photothermal signals, ensuring production only on demand when required by the wound, thus avoiding potential risks associated with overexpression(Guggenbichler et al., 2016).
3. Lab Safety
Laboratory safety is a crucial component of the iGEM project. We implemented various measures to ensure the biosafety of the iGEM lab for all participants.
3.1 Safety Regulations
All wet lab experiments were conducted under the MOE Key Laboratory of Evolution & Marine Biodiversity at Ocean University of China (OUC). According to university regulations, all experimental procedures complied with the "Ocean University of China Laboratory Biosafety Management Measures." All our wet lab work was performed in laboratories meeting Biosafety Level 1 (BSL-1) standards and strictly adhered to the following regulations:
All team members studied and strictly adhered to the "Biosecurity Law of the People's Republic of China" and the university's laboratory safety management regulations. They passed the laboratory safety access exam, obtaining the qualification to perform experimental operations.
Img3&4: the lab safety files in the official website, and we learn many lab managements in these websites.
3.2 Safety Education
Before conducting wet lab experiments, our PIs and instructors provided us with usage specifications and detailed instructions for many lab’s equipment, particularly emphasizing precautions for the autoclave and biological safety cabinet. Through their guidance, we greatly enhanced our understanding of the safety regulations that must be followed in the lab and the safety considerations required during experiments.
Img5. The lyophilizer we used, and the detailed instructions
3.3 Routine Lab Management
To remind everyone to pay attention to safety during experiments, numerous safety signs were posted in the laboratory, and instructions for specific equipment were displayed on the relevant instruments. During our team's regular meetings, we also discussed issues encountered during experiments, such as the classification of general waste vs. laboratory waste, and protocols for water changes after autoclaving discarded materials. This ongoing engagement deepened our understanding of laboratory safety regulations.
4. Environmental Safety
We prospectively considered the safety issues that might arise from the future application and disposal of the product:
Environmental Friendliness
Biodegradable:The main component of the hydrogel, chitosan, is derived from renewable natural resources like shrimp and crab shells. It is fully biodegradable after use and will not cause environmental residue.
Green Production:The therapeutic proteins are biosynthesized directly by yeast cells. Compared to traditional chemical pharmaceutical production, this avoids the use of large amounts of toxic reagents and energy consumption, making the process more environmentally friendly.
Waste Disposal:Used dressings are treated as medical waste and can be processed through standard incineration or autoclaving procedures, ensuring the complete inactivation of any engineered yeast within them.
5. Conclusion
In summary, the Yeast Medics project has undergone comprehensive consideration and design across multiple dimensions including biosafety, design safety, operational safety, and environmental safety. We are not only committed to developing an effective new solution for diabetic wound treatment but also consistently prioritize responsible research and innovation, ensuring that our technological development path is safe, reliable, and sustainable.
References
[1] Arnesen, J. A., & Borodina, I. (2022). Engineering of Yarrowia lipolytica for terpenoid production. Metabolic Engineering Communications, 15, e00213. https://doi.org/10.1016/j.mec.2022.e00213
[2] Guggenbichler, F., Büttner, C., Rudolph, W., Zimmermann, K., Gunzer, F., & Pöhlmann, C. (2016). Design of a covalently linked human interleukin-10 fusion protein and its secretory expression in Escherichia coli. Applied Microbiology and Biotechnology, 100(24), 10479–10493. https://doi.org/10.1007/s00253-016-7667-5
[3] Park, Y.-K., & Ledesma-Amaro, R. (2023). What makes Yarrowia lipolytica well suited for industry? Trends in Biotechnology, 41(2), 242–254. https://doi.org/10.1016/j.tibtech.2022.07.006
[4] Ocean University of China. (2019, October 14). Lab safety-safety knowledge. Ocean University of China_State-Owned Assets and Laboratory Management Office; Ocean University of China. http://gzc.ouc.edu.cn/aqzs_20548/list.htm
[5] Sun, M., Wang, T., Pang, J., Chen, X., & Liu, Y. (2020). Hydroxybutyl Chitosan Centered Biocomposites for Potential Curative Applications: A Critical Review. Biomacromolecules, 21(4), 1351–167. https://doi.org/10.1021/acs.biomac.0c00071
[6] Yue, L., Chi, Z., Wang, L., Liu, J., Madzak, C., Li, J., & Wang, X. (2008). Construction of a new plasmid for surface display on cells of Yarrowia lipolytica. Journal of Microbiological Methods, 72(2), 116–123. https://doi.org/10.1016/j.mimet.2007.11.011
