Team:GA State SW Jiaotong/Design

Collaboration

Design

For decades, close attention has been paid to treating people worldwide who are suffering from alopecia and the mental stress caused by it. Recent researches have revealed the fundamental role of Gas6 and EGF proteins in reactivating human follicle cell growth. Gas6, the growth arrest-specific protein, can efficiently reactivate hair follicle stem cells that proceed into the dormant phase under chronic stress.[1] On the other hand, epidermal growth factor (EGF) can effectively stimulate hair follicle cell growth and improve the microenvironment and blood supply of the scalp.[2]

Gas6 and EGF expression

Based on our survey results, we learned that many people suffer from hair loss caused by chronic stress and androgen disorder and have difficulty finding an effective drug or a suitable treatment method. Consequently, we came up with the idea of heterologously expressing Gas6 and EGF in yeast and bacteria and creating a product to treat androgenetic alopecia and stress alopecia.

Since Gas6 is a mammalian protein that has never been expressed heterologously before, we chose Pichia pastoris GS115, a eukaryotic cell line, to express the anthropogenic protein. By inserting the Gas6 coding sequence and an alpha secretion factor onto the pPICZäA vector, we hope to purify the Gas6 proteins from the yeast. In addition, considering that the mass production system using E. coli and T7 promoter to express proteins is very mature, we also hope to use pNW33N-T7-Gas6 and pNW33N-T7-EGF plasmids to overexpress Gas6 and EGF in E. coli.

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Fig1.Gas6 and EGF expression

If the protein expression of Gas6 and EGF in P. pastoris or E. coli works properly, we will get the proteins purified and mix the product with nanoscale emulsion. Then, combining the product with micro-needle technology, the drug will be delivered efficiently to the location of human follicle stem cells and reactivate the cells from the dormant phase.

However, since preserving the protein products at home is difficult, customers may need to visit professional institutes such as hospitals or beauty salons to receive treatment. In addition, since the injection process may be intimidating to customers, we came up with another customer-friendly, but bold design——use engineered Bacillus subtilis to express Gas6 and EGF directly on the human scalp! In addition to the bacteria usually found on the human scalp, an engineered strain of B. subtilis transformed with pNW33N-P43-Gas6 and pNW33N-P43-EGF will be added to the human scalp to steadily express the target proteins.

Considering the safety and ethical issues that this will be used by the everyday person, a suicide switch design is required for the living-cell system. During the communication with Tongji_China and ShanghaiTech_China teams, we finally established a toxin-antitoxin suicide system using a dual regulation design provided by ShanghaiTech University. Using hok and sok proteins as the toxin and antitoxin components, we will regulate the transcription using two promoters, Ptrp and Plac. The transcription of the hok gene will be blocked by excessive tryptophan under the effect of Ptrp, while the lactose operon, which controls the transcription of sok, is induced by lactose (or IPTG).

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Fig 2. Schematic diagram of toxin-antitoxin (Hok/Sok) mechanism.

Therefore, in the presence of tryptophan and lactose, the transcription of the hok gene will be inhibited, while the hok gene is activated; and the bacteria will survive. Under environmental changes where tryptophan and lactose are missed, the toxin gene, hok, will be expressed while the transcription of sok is inhibited. This will result in the death of the bacteria due to the depolarization of the cell membrane caused by hok. [3]

Finally, the end product is a result of combining our engineered B. subtilis solution with hydrogel. An essential oil product paired with shampoo for following use is expected.

Source

1.Choi, S., Zhang, B., Ma, S., Gonzalez-Celeiro, M., Stein, D., Jin, X., Kim, S. T., Kang, Y. L., Besnard, A., Rezza, A., Grisanti, L., Buenrostro, J. D., Rendl, M., Nahrendorf, M., Sahay, A., & Hsu, Y. C. (2021). Corticosterone inhibits GAS6 to govern hair follicle stem-cell quiescence. Nature, 592(7854), 428–432. https://doi.org/10.1038/s41586-021-03417-2

2.Gao, H. C. & Liu, J. Q. (2018). Research advance of concentrated growth factors in treatment of androgenetic hair loss. Chin J Clin Lab Mgt, 6(4), 193-198. https://doi.org/10.3877/cma.j.issn.2095-5820.2018.04.001

3.Hayes, F. (2003). Toxins-antitoxins: plasmid maintenance, programmed cell death, and cell cycle arrest. Science, 301 (5639): 1496–9. https://doi.org/10.1126/science.1088157




Collaboration