Team:Estonia TUIT/Safety
We carried out the experimental work in a Biosafety Level 1 laboratory with organisms belonging to the white list.
Training and supervision
Before starting the laboratory work, all members of our team had successfully passed the safety and security training. While working, our lab members are constantly guided and supervised by our instructors. We strictly follow the norms and regulations of the Institute of Technology for experimental work.
Organisms used in our experimental work
We use non-pathogenic organisms (Saccharomyces cerevisiae and Escherichia coli) that belong to the white list in order to make the experimental work and subsequent implementation safer. We follow biohazard waste disposal rules to ensure that no modified cells are released from the lab. If the strain developed will later be used in industrial bioreactors, then we need to further consider the possible risks to humans and the environment.
Safety of the final product
Our project develops an innovative oral hygiene product that is based on an engineered protease – thus, the safety and security questions are central in our final product and the production process.
We also chose trypsin as a candidate starting protease for our experiments, as it is inactivated at low pH, thus it will lose activity in the gastrointestinal tract, increasing the safety of the product. We have started with the analysis of the trypsin safety data sheet. According to Safc Biosciences Material Safety data sheet, these are general instructions when working with trypsin and in case of:
We have started with the analysis of the trypsin safety data sheet. According to Safc Biosciences Material Safety data sheet, these are general instructions when working with trypsin and in case of:
a) Trypsin contact with eyes: it is obligatory to flush thoroughly with water and contact a physician (Trypsin-EDTA Solution 1X, 2006);
b) Trypsin contact with skin: wash the affected area with soap and water. If irritation will occur, contact a physician (Trypsin-EDTA Solution 1X, 2006);
c) Accidental swallowing of trypsin: rinse the mouth with fresh water and contact a physician (Trypsin-EDTA Solution 1X, 2006);
d) Accidental inhalation of trypsin: take the affected person to fresh air and contact a physician (Trypsin-EDTA Solution 1X, 2006). If breathing becomes difficult, give oxygen. If breathing stops, give artificial respiration (Trypsin-EDTA Solution 1X, 2006).
Importantly, the Atlantic cod trypsin has been previously used in biomedical studies and is reported as non-toxic (Gudmundsdóttir et al., 2013). In modeling the SALSA cleavage, the Atlantic cod trypsin showed promising efficiency (please, refer to the Modelling page). Trypsin’s normal working pH level, which is in a range 7-9 units, (Lam et al., 2012) differs significantly from the stomach acidic pH, which range is 1.0-2.5 units (Evans et al., 1988). Trypsin protease gets fully inactivated at pH 4 and below (Review of Proteins & Enzymes), thus, the protease will not be active in the gastrointestinal tract. It was shown that after incubation in the intestinal juice for 30 minutes, only <5% of the enzyme can be active (Wohlman et al., 2016). There is a difference between the neutral environment (pH 7) of the esophagus (Tutuian & Castell, 2006) and acidic pH in the stomach.
As an additional safety consideration to experimentally examine the possibility of some amount of the enzyme being absorbed in the esophagus, we can implement an addition of non-toxic irreversible trypsin inhibitor, named α1-antitrypsin (Serres & Blanco, 2014). α1-antitrypsin fully inactivates the enzyme and reduces the risk of cleaving other proteins, as enzyme activity is limited to a short time period of staying in the mouth.
The potential toxicity of the engineered protease will need further examination
Also, the presence of some allergens and insufficient purity are possible sources of risks of our final product. Since our final product contains only the purified protease in a cell free solution, there is a negligible risk of the GMO being released from the production process. To ensure that the genetically modified yeast used for the production of the protease will not be released from the lab, we will introduce a multistep kill switch to the yeast. This provides an additional security level to guarantee that the GMO will not survive in a natural environment.
Gudmundsdóttir, Á., Hilmarsson, H., & Stefansson, B. (2013, February 28). Potential use of Atlantic cod trypsin in Biomedicine. BioMed Research International. Retrieved October 16, 2021, from https://www.hindawi.com/journals/bmri/2013/749078/. de Serres, F., & Blanco, I. (2014). Role of alpha-1 antitrypsin in human health and disease. Journal of Internal Medicine, 276(4), 311–335. https://doi.org/10.1111/JOIM.12239 Evans, D. F., Pye, G., Bramley, R., Clark, A. G., Dyson, T. J., & Hardcastle, J. D. (1988). Measurement of gastrointestinal pH profiles in normal ambulant human subjects. Gut, 29(8), 1035. https://doi.org/10.1136/GUT.29.8.1035 Lam, M. P. Y., Lau, E., Liu, X., Li, J., & Chu, I. K. (2012). Sample Preparation for Glycoproteins. Comprehensive Sampling and Sample Preparation, 3, 307–322. https://doi.org/10.1016/B978-0-12-381373-2.00085-5 Trypsin-EDTA Solution 1X. (2006). www.safcbiosciences.com Tutuian, R., & Castell, D. O. (2006). Gastroesophageal reflux monitoring: pH and impedance. GI Motility Online, Published Online: 16 May 2006; | Doi:10.1038/Gimo31. https://doi.org/10.1038/GIMO31 Wohlman, A., Kabacoff, B. L., & Avakian, S. (2016). Comparative Stability of Trypsin and Chymotrypsin in Human Intestinal Juice: Https://Doi.Org/10.3181/00379727-109-27092, 109(1), 26–28. https://doi.org/10.3181/00379727-109-27092
