Team:St Andrews/Implementation

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Implementation

As discussed in the ‘Laboratory Research’ section of our wiki, during the six weeks (5th July – 13th August) in which our St Andrews 2021 iGEM team conducted laboratory experiments, our team only managed to successfully clone one (out of four total) genes of the shinorine-producing pathway into its respective plasmid via Gibson Assembly. Clearly, much more lab work must be carried out before our final sunscreen product is ready for implementation in the real world. Below is an outline of the next steps that our team would have to take in order to start selling Shinescreen (our reef-safe sunscreen) to our target customers. First, our wetlab team would have to successfully clone all the genes involved in the shinorine-producing pathway (DHQS, O-MT, ATPG, and NRPS) into plasmids, transform these plasmids into BL21 E. coli expression cells, and finally confirm the overexpression of each of the four enzymes involved in producing shinorine. Our team would then have to confirm that the BL21 E. coli bacteria would be capable of synthetically producing shinorine in significant enough quantities to guarantee UV-A protection.

Secondly, our iGEM team would have to re-design our killswitch (since the kill-switch parts designed by last year’s team proved too difficult for manufacturing by IDT), subsequently implement the kill-switch into the same plasmids that contain our Shinogen parts, and then also transform these plasmids into BL21 E. coli expression cells. These vital steps would ensure the biosafety of our organism, since the killswitch would protect against horizontal gene transfer as well as the escape of our bacteria into the surrounding environment. Our team would then have to carry out multiple laboratory tests to ensure that our bacteria could only survive when they are both exposed to sunlight and have a plentiful supply of glucose. In other words, if either one (or both) of these two conditions were not fulfilled, the bacteria would be killed due to the action of the killswitch: these strict conditions would ensure that the bacteria would not present any health or environmental concerns when developed as a sunscreen for use by consumers.

Next, our iGEM team would have to repeat the above steps, but instead transforming the plasmids containing our shinogen and thanogen parts into Nissle 1917 E. coli cells (as opposed to BL21 E. coli cells). The use of Nissle 1917 E. coli cells would be crucial for our project, since this strain of bacteria is non-disease causing, FDA-approved, and probiotic, meaning that the application of this synthetically engineered organism onto human skin would theoretically be a completely safe procedure. Of course, our team would have to conduct experimental tests (both on humans, and within the marine environment) to confirm that these synthetically engineered bacteria do not cause any side-effects to human health, nor harm to aquatic ecosystems and marine life (this testing stage is of course entirely theoretical, and would take place outside the scope of iGEM).

Alongside this stage, our team would have to explore the incorporation of additional mycosporine-like amino acids (MAAs) into our sunscreen. While shinorine does effectively confer protection against UV light, it only does so within the 280 nm-360 nm range, with maximal absorption occurring at 320nm – 340 nm (this corresponds to the UV-A2 range, (NAGASE, 2018)). Hence, for our Shinescreen product to be a viable competitor within the sunscreen market, our bacteria would have to synthetically overproduce additional (naturally-occurring, and reef-safe) molecules that absorb UV light within other wavelength ranges, in order that our sunscreen product can confer protection against the total UVA – UVB range.

Our team would also have to develop a formula in which to store the bacteria. This formula would have to contain a starting medium of glucose - this is a primary feedstock for continued metabolism in the cells, as well as a feedstock for the thanogen (iGEM St Andrews, 2020). In order that the shelf-life of our sunscreen is maintained, our team would also have to test the most appropriate method of storing our bacteria. These methods would likely include microencapsulation (there are many different microencapsulation technologies to choose from (Martín et al., 2015)), airdrying, or freeze-drying. In the latter two methods, compatible solutes (such as trehalose) would be added to the cream in order to help preserve the bacteria once dried (Louis, Trüper, and Galinski, 1994). Our team would additionally try to ensure that our formula is transparent, so that our sunscreen will be adaptable for use on all skin tones. This feature is very important to us, as we want inclusivity and diversity to be a prominent feature of our brand.

Lastly, our team would design and produce eco-friendly packaging in which to store our sunscreen product. The packaging we would use to store our sunscreen would be minimal, likely only consisting of a sunscreen bottle. This would further ensure the sustainability of Shinescreen, as the lack of plastic packaging would further benefit the marine environment (plastic pollution is a very widespread problem affecting aquatic organisms (Marine Plastics, 2018)).

References

2018. Marine Plastics. [pdf] Gland: IUCN. Available at: https://www.iucn.org/sites/dev/files/marine_plastics_issues_brief_final_0.pdf

2018. Shinorine - A natural, soluble anti-photoageing ingredient -. [PDF] NAGASE & CO., LTD. Available here iGEM St Andrews 2020 (2020) Implementation, Shinescreen. Available here

Louis, P., Trüper, H. G. and Galinski, E. A. (1994) ‘Survival of Eschericia coli during drying and storage in the presence of compatible solutes’, Applied Microbiology and Biotechnology, 41(6), pp. 684-688. DOI: 10.1007/BF00167285.

Martín, M., Lara-Villoslada, F., Ruiz, M. and Morales, M., 2015. Microencapsulation of bacteria: A review of different technologies and their impact on the probiotic effects. Innovative Food Science & Emerging Technologies, 27, pp.15-25. DOI: 10.1016/j.ifset.2014.09.010