It is important to address the public’s and the lawmakers’ concerns when dealing with genetically modified organisms. Furthermore, the wellbeing of each team member must be ensured. For these reasons different measures were taken, varying from the choice of the working organisms to implementation of a kill switch.

Chassis

When working with food products, the choice of a suitable chassis is crucial for the safety of the consumers. Hence, we put considerate thoughts into deciding on what organism is best for our biosensor and finally chose a strain of the fungi Saccharomyces cerevisiae.
Having been widely used in the production of baked goods and fermented alcoholic beverages for many centuries⁷, S. cerevisiae not only belongs to the GRAS (generally regarded as safe) organisms, but it was also the first eukaryote to have its genome fully sequenced why it has served as a model organism ever since ⁴. Well characterized is also its pheromone response pathway, which is well suited for and has been previously used to be reengineered to a biosensor ⁸. Moreover, the strain that we are using (AM254) has been optimised towards the use as biosensor by the implementation of five mutations in the causative genes of the pheromone response pathway.

Parts

The function of a biosensor is to turn a signal input into a measurable output. In the course of our project and in line with the responses that we got from the outside, we have decided that this output should be in the form of a colour. Working on a proof of concept, this colour change is currently achieved by the expression of the fluorescent protein sfGFP as this is widely used and easily measurable under a fluorescent microscope. However, in the future we will need compounds that are visible to the naked eye and known to be harmless to the human body. Therefore, we have considered the two natural pigments betalain and indigoidine as potential colour outputs. Betalains can either show yellow to orange or reddish to violet colours and are mostly known for giving beets their significant colour. Indigoidine on the other hand is naturally produced by some bacteria and results in a bright blue colour. It has been listed as promising natural alternative as food dye in several publications^{9, 6}.

In addition to the reporter compounds, our yeast strain will also produce the two proteins Bax and BI-1 that are needed for the implemented killswitch (see below). Bax is a pro-apoptotic regulator. When activated via apoptosis inducing signals Bax associates with mitochondria and promotes the release of cytochrome C and other apoptotic protein, leading to activation of caspases and cell death⁵. If Ovulaid chewing gum were realized exposure of Bax to humans is not expected to be a concern. To enter human cells it would need an active transporter, and even if it were internalized in cells, it would need further apoptotic signalling. If ingested via chewing gum, Bax would be degraded into amino acids or small peptides as soon as it enters the stomach¹.

Kill Switch

To address the safety of our biosensor as well as the strict conditions and fear of public that come with the usage of GMOs, we have implemented a killswitch. The idea and principle for it comes from the iGEM Team of NAU China 2017. Its principle is rather simple and based on the agonistic correlation of the mammalian derived toxin Bax and its antitoxin BI-1. Both of these proteins belong to the Bcl-2 family, whose members either inhibit or promote cell apoptosis².
While the toxin is constitutively produced, the antitoxin is induced by a compound that is present in the chewing gum. As a result, the toxin is balanced out by the antitoxin when the yeast is inside the gum. As soon as it escapes into the environment though, the antitoxin isn’t produced anymore and the yeast will die.

References

1. Berg, J. M., Tymoczko, J. L., & Stryer, L. (2002). Proteins Are Degraded to Amino Acids. In Biochemistry (5th Edition). W H Freeman.
2. Boise, L. H., Gonzblez-Garcia, M., Postema, C. E., Ding, L., Lindsten, T., Turka, L. A., … Thompson, C. B. (1993). bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell, 74, 597–606.
3. Bolduc, N., Mario, O., Pitre, F., & Brisson, L. F. (o. J.). Molecular characterization of two plant BI-1 homologues which suppress Bax-induced apoptosis in human 293 cells. https://doi.org/10.1007/s00425-002-0879-1
4. Goffeau, A., Barrell, B. G., Bussey, H., Davis, R. W., Dujon, B., Feldmann, H., … Oliver, S. G. (1995). Life with 6000 genes. Science, 274(5287), 546–567. https://doi.org/10.1126/science.7542800
5. Hsu YT, Wolter KG, Youle RJ (April 1997). "Cytosol-to-membrane redistribution of Bax and Bcl-X(L) during apoptosis". Proc. Natl. Acad. Sci. U.S.A. 94 (8): 3668–72.
6. Khajuria, R. (2003). Natural pigments: an alternative to synthetic food colorants. In Technologies in Food Processing (S. 155–178).
7. Legras, J. L., Merdinoglu, D., Cornuet, J. M., & Karst, F. (2007). Bread, beer and wine: Saccharomyces cerevisiae diversity reflects human history. Molecular Ecology, 16(10), 2091–2102. https://doi.org/10.1111/j.1365-294X.2007.03266.x
8. Shaw, W. M., Yamauchi, H., Mead, J., Gowers, G.-O. F., Öling, D., Larsson, N., … Ellis, T. (2018). Engineering a model cell for rational tuning of GPCR signaling. bioRxiv, 1–15. https://doi.org/10.1101/390559
9. Yangilar, F., & Yildiz, P. O. (2016). The final development related microbial pigments and the application in food industry. Journal of Science and Technology, 9(1), 118–142. https://doi.org/10.18185/eufbed.55880