Team:GA State SW Jiaotong/Engineering
Engineering design—build—test—learn
Like all engineering, Hnoss utilizes scientific principles to design and build the engineering bacteria containing DNA encoding for protein Gas6 and EGF that help alleviate the hair loss burden. Hnoss aims to produce a new application for the real world, based on a successful engineering cycle through "design-build-test-learn."
Fig1.Engineering cycle:design-build-test-learn
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]
Our survey results taught 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。
Fig2.whether you are troubled by hair loss
Fig3.Severity of hair loss
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.
Build
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 purify the Gas6 proteins from the yeast and build the new parts BBa_K3842006. In addition, considering that the mass production system using E. coli and T7 promoter to express proteins is very mature, we also build new parts BBa_K3842015,BBa_K3842010 that use pNW33N-T7-Gas6 and pNW33N-T7-EGF plasmids to overexpress Gas6 and EGF in E. coli.
Test
Plasmids Transformation
At very beginning, Gas6 coding sequence was amplified by PCR with primer Gas6+signal_top and Gas6+signal_bottom. Then, Gas6 coding sequence and pNW33N+T7 were cut by HindIII and SacI. After that, two sequence were linked by ligation. Final plasmid was transformed into WB600 and single colonies were obtained from overnight incubation on LB+ CM plate.
Fig4. Transformation results of Pichia Pastoris transformed with pNW33N-Gas6. The control group: pNW33N+Gas6+Lac promotor (LB). The experimental group: pNW33N+Gas6+Lac promotor (LB+CM). Many single colonies were obtained after overnight incubation according to the experimental group.
Electrophoresis
We did a PCR and obtained the electrophoresis result to test whether the engineering bacteria is existing. According to the Electrophoresis result, sok gene had same size as expected. However, the length of hok gene was not consistant with the expected.
Fig5.Electrophoresis result. Line1: 100bp DNA Ladder. Line2: Hok 09/01. Line3: Sok 09/03. Line4: Sok 09/29. Line5: Gas6 09/03. Line6: Gas6 09/01. Line7: pNW33N-T7-Gas6. Line8: TRP 09/08. Line9: TRP 09/03. Line10: 1kb DNA Ladder.
Testify the Existence of target protein
SDS-PAGE and Western-blot are used to testify the existence of Gas6 protein. However, the result of Western-blot indicated that there was no Gas6 protein in any of our sample. Thus, we began to learn from our failure.
Fig6. Western blotting result. The first line and the last line were protein ladders.
Between them, no bands exist.
Learn
After a series of experiments, we consulted with Dr. Kyu, a Research Scientist at Georgia State University, about the reason for our lack of positive results. Through our conversations, we came to realize some of the shortcomings in the operation of our experiment.
Dr. Kyu pointed out that there might be plasmid mutations after extraction, so we should sequence it before starting the follow-up experiment. Moreover, he suggested that we isolate and streak, and cultivate the single colony we chose to carry out for the PCR amplification to guarantee that all subsequent experimental results are derived from the same colony. Besides, we were supposed to use promoters with a high-copy number. We also ignore that the proteins we selected were human-derived and should be a post-translational modification.
We summed up the problem with the help of Dr. Kyu, and we tried to improve and redesign our experiment to do more experiments.
