Safe Project Design and Execution
The purpose of Oviita is to improve the health and wellbeing of those affected by worldwide Vitamin A deficiency. This goal would be entirely pointless if we did not adequately ensure the safety of the solution itself and our own communities. As with any medical intervention, the first principle must always be do no harm. Therefore, we have strived to consider every aspect of safety both of our future product, and our our practices in the workplace.
IN PROJECT DESIGN
Investigations into food safety
Yarrowia lipolytica has been evaluated and approved for safe consumption as an edible nutritional yeast by a variety of sources. It was approved by the European Food Safety Authority in 2019 for consumption of whole-cell biomass after being heat-killed. Their findings concluded that there were no nutritional or toxicological concerns for human dietary use, and granted permission to produce Yarrowia cell mass as a safe edible supplement. Their recommendations suggested a maximum of 3g per day of dried cell biomass for children from 3 years to 10 years old, and 6 g/day afterwords. Additionally, Y. lipolytica is already a common component of dairy and meat products, particularly sausages. It is part of the desirable flora of common cheeses including Gouda, Picante, Apulian, Camembert, blue-veined, Cheddar, Brie, and Feta (Zinjarde, 2013), where these yeast contribute to the overall flavour.
A study by Groenewald et. al (2014) of potential pathogenicity of Y lipolytica concluded that the yeast could be considered on an equivalent level of non-pathogenicity as S. cerevisiae, with only a few rare examples of opportunistic infection in severely immunocompromised individuals. It is generally regarded as non-pathogenic (Zinjarde, 2013), is common and ubiquitous in the environment (EPA, 2019), and has been granted Generally Recognized As Safe (GRAS) status by the U.S. Food and Drug Administration (USDA) (Zieniuk 2019). However, Oviita should nonetheless be avoided by individuals with compromised immune systems as well as children under 3 years old.
Additionally, studies on recombinant beta-carotene produced by Y. lipolytica found that it did not produce effects any different from commercial beta-carotene, and shared a similar safety profile regardless of the different source. (Grenfell-Lee et al., 2013)
Finally, thymol is also regarded as a safe food ingredient. It is classified by the FDA as a food for human consumption as well as a food additive and flavouring ingredient, and possesses GRAS designation (EPA, 1993). Naturally occurring in aromatic plants especially thyme, it is widely consumed by humans. A review of its status by the EPA in 2009 concluded that thymol is a normal part of the diet and has minimal toxic effects in mammals, and is therefore an acceptable food additive. Future experimental design will seek to quantify the thymol output of Oviita yeast under various field conditions to ensure that thymol concentrations are both effective and safe.
As with any biotechnology project, safety in terms of effective biocontainment must be kept in mind. Especially when considering the eventual goal of community implementation for our project, we need a biocontainment system to prevent the survival of our yeast strains outside of their intended growth vessel. We developed a sustainable and affordable biosafety system where two yeast strains will be co-cultured together in a syntrophic community. These strains will each be auxotrophic for a molecule the other strain has been modified to overproduce. This ensures that the two strains are co-dependent, reducing the chances of environmental escape. The organism can only proliferate if both strains are present in sufficient quantities to support each other’s growth. Ultimately, this novel method of co-dependent auxotrophy provides an easily implementable biocontainment strategy to prevent the escape of genetically engineered organisms. You can learn more about our biocontainment system here.
GENERAL LAB SAFETY
Ensuring a safe lab environment
Before participating in the lab, all members of our wet-lab team completed a set of essential safety training courses. These courses provided training in hazard and risk assessment, reporting incidents and near misses, occupational health and safety, laboratory safety, WHMIS, spill response, and biosafety. This training allowed us to work safely, and responsibly manage any concerns or risks that could arise during routine lab work. We adhered at all times to the guidelines of the University of Calgary Biosafety Committee, which outlines good biological laboratory practices to ensure that the university research environment can remain safe and responsible. All of our members are committed to the safety of our team and community, making lab safety a priority.
As a team
Covid-19 has presented a unique set of challenges and risks to the scientific community. Despite this, our team has remained committed to caring for the safety of each other and those around us. Due to this, like so many iGEM teams, we have radically restructured our time and practices to allow for social distancing and risk management. Our school semester was cut short due to the closure of the university. As a result of this, the last portion of our synthetic biology class MDSC 507 was completed virtually. As the winter semester transitioned to the summer, we remained at home, and developed plans to complete our work digitally for the summer. Zoom and various organizational softwares have allowed us to complete dry lab and wet lab theoretical work without access to the lab space all summer. We met with other teams, attended events, and met all of our HP contacts through virtual means as well.
In Wet Lab
During the fall semester, with Covid-19 cases reasonably managed in our region, we were able to return to the lab part-time, with careful precautions to minimize risks. All members wore masks at all times. Additionally, only six members and a supervisor were ever in the lab at one time, and respectful 2 metre social distancing was maintained. Our hours in the lab were also limited to just 6 hours per week at arranged times, with all work that could be done elsewhere, including meetings and planning, being conducted virtually.
In Dry Lab
Due to the nature of dry lab projects, we were able to finish them with no in-person meetings. Collaboration on projects involving programming were done through online platforms like Zoom and Slack. When building our hardware tools two members met in-person while wearing masks at all times and maintaining a 2-meter distance as much as possible.
Environmental Protection Agency (EPA). (1993, September). US EPA - Pesticides - Fact Sheet for Thymol. United States of America EPA. https://www3.epa.gov/pesticides/chem_search/reg_actions/reregistration/fs_PC-080402_1-Sep-93.pdf
Environmental Protection Agency (EPA). (2009). Thymol; Exemption from the Requirement of a Tolerance (74 FR 12613, pp. 12613-12617, Rep.). United States of America EPA. doi:https://www.federalregister.gov/documents/2009/03/25/E9-6262/thymol-exemption-from-the-requirement-of-a-tolerance
FSA NDA Panel (EFSA Panel on Nutrition, Novel Foods and Food Allergens), Turck, D, Castenmiller, J, de Henauw, S, Hirsch‐Ernst, K‐I, Kearney, J, Maciuk, A, Mangelsdorf, I, McArdle, HJ, Naska, A, Pelaez, C, Pentieva, K, Siani, A, Thies, F, Tsabouri, S, Vinceti, M, Cubadda, F, Engel, K‐H, Frenzel, T, Heinonen, M, Marchelli, R, Neuhäuser‐Berthold, M, Pöting, A, Poulsen, M, Sanz, Y, Schlatter, JR, van Loveren, H, Ackerl, R and Knutsen, HK. (2019). Scientific Opinion on the safety of Yarrowia lipolytica yeast biomass as a novel food pursuant to Regulation (EU) 2015/2283. EFSA Journal 2019;17(2):5594, 12 pp. https://doi.org/10.2903/j.efsa.2019.5594
Grenfell-Lee, D., Zeller, S., Renato, C., & Pucaj, K. (2014). The safety of β-carotene from Yarrowia lipolytica. Food and Chemical Toxicology, 65, 1-11. doi:https://doi.org/10.1016/j.fct.2013.12.010
World Health Organization (WHO). (2009). Global prevalence of vitamin A deficiency in populations at risk. WHO Global Database on Vitamin A Deficiency. https://apps.who.int/iris/bitstream/handle/10665/44110/9789241598019_eng.pdf?sequence=1
Zieniuk, B., Fabiszewska, A. Yarrowia lipolytica: a beneficious yeast in biotechnology as a rare opportunistic fungal pathogen: a minireview. World J Microbiol Biotechnol 35, 10 (2019). https://doi.org/10.1007/s11274-018-2583-8
Zinjarde, S. S. (2014). Food-related applications of Yarrowia lipolytica. Food Chemistry, 152, 1-10. doi:https://doi.org/10.1016/j.foodchem.2013.11.117