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Team:Montgomery Cougars NJUSA/Project/TheProcessofSelectingtheGene
From 2014hs.igem.org
Selecting the Gene
Initially, we planned to create a cocktail of enzymes that would break down components of sebum (triglyceride oils, wax, squalene, and metabolites of fat-producing cells). However, as a first year team, we decided that choosing one enzyme would be the most practical.
| Enzyme | Gene |
|---|---|
| Triglyceride Lipase: lipases that hydrolyse ester linkages of triglycerides | PNPLA2 gene provides instructions for making an enzyme called adipose triglyceride lipase |
| Triacylglycerol Lipase: the fat-splitting enzyme in pancreatic juice; it hydrolyzes triacylglycerol to produce a diacylglycerol and a fatty acid anion
triacylglycerol + H2O diacylglycerol + a carboxylate | LIPC gene encodes hepatic triglyceride lipase |
| Squalene epoxidase: an enzyme released by the dermatophyte fungi to break down Squalene. | SQLE gene encodes squalene epoxidase |
| HMG CoA Reductase: an enzyme that catalyzes the production of mevalonate from HMG CoA. It is a rate controlling enzyme in the mevalonate pathway that regulates the synthesis of cholesterol and other isoprenoids. | HMGCR gene encodes HMG CoA Reductase |
| Lipoprotein Lipase: water soluble enzyme that hydrolyzes triglycerides into two fatty acids. | LPL gene encodes Lipoprotein Lipase |
In the beginning, we chose to use lipase as the enzyme to eliminate the bacterial food source. Because sebum is composed of 57.5% triglycerides and fatty acids, we decided that lipase which breaks down triglycerides into free fatty acids would be the optimal protein. However, through further research, we learned that when lipase converts triglycerides to free fatty acids, it only exacerbates the inflammatory response. Free fatty acids increase clumping of bacteria, which in turn facilitates the colonization in the duct. When inflammatory mediators penetrate the skin, an inflammatory reaction is initiated. P. acnes lipase (GehA, glycerol-ester hydrolase A) is in fact a virulence factor involved in the pathogenesis of acne. In Shuichi Higaki’s research paper, Higaki asserts that a lipase inhibitor can help treat acne. We contemplated the idea of trying to find a lipase inhibitor; however, few of the inhibitors are proteins. We contemplated using vitamin A, but it proved too complicated with too little standardization of use in accordance to the iGEM registry. In another approach, we thought we could break down the fatty acids before they were absorbed into a cell. The challenge was that acne vulgaris is a multi-factorial disease.
| Target | Notes |
|---|---|
| Comedones | IL-1 alpha (causes hyperkeratosis). IL-1 alpha receptor antagonist? |
| Androgens | But, association of acne + high androgen concentration is less consistent
Not as well studied Correlation not clear and is possibly dangerous/harmful to female users |
| Sebum(change in qualitative change in sebum lipids induce alteration of keratinocyte differentiation and induce IL-1 secretion) | Target the fatty acids
The lipolysis of lipids comes from a signal transduction pathway initiated with epinephrine, norepinephrine, growth hormone, testosterone etc that triggers a GPCR that initiates lipolysis. There’s this protein called serum albumin that acts as a carrier of fatty acids. If they’re carried away into the bloodstream, they cannot be fed on by bacteria. Get this, there’s a gene for it too :) Target the squalene Target the squalene oxide Target the sebaceous linoleic acid (more linoleic acid=less sebum production) |
| P. acnes | Use of antibiotics (perhaps we could synthesize one of them) |
| Vitamin A→(hypothesis that hyperkeratosis is caused by local deficiency of vitamin A) | It doesn’t look promising because vitamin A is an ingestible molecule found in plants and beta carotenes like carrots |
| Fatty acids | Potential enzyme: Serum Albumin, carries fatty acids and hemin through human respiratory system. Coded by ALB gene. |
While we had all of these possibilities, many were completely unfeasible and unrealistic to try to accomplish. In the end, we decided on Medium Chain Alcohol and Aldehyde dehydrogenase. See our final decision.
