Difference between revisions of "Team:UMaryland/Description"

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Lutein is a dietary supplement used as both a treatment and preventative measure for Age-Related Macular Degeneration. Our primary research focus is to combat AMD through lutein production. Lutein is currently extracted from the petals of marigolds, but we hope to create a more cost-effective and efficient way of synthesizing this carotenoid via bacterial pathways. Our goal is to optimize the production of lutein through the careful manipulation of promoters for various enzymes, not native to E. Coli, in the lutein pathway. This pathway begins with lycopene, which is then converted to alpha-carotene with the addition of epsilon- and then beta-cyclase. Alpha-carotene is then converted to lutein through epsilon- and beta-hydroxylase. In order to ensure that we are producing alpha carotene instead of beta carotene, we will be manipulating the regulation of epsilon and beta cyclase by testing various promoter pairings. We will then use this data to help model the synthetic pathway and hopefully create a user friendly tool to help iGEM teams in the future model synthetic projects.
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Advanced Macular Degeneration is the current leading cause of blindness and vision loss in people aged 65 and over. 1.75 million people in the US are affected with AMD, and that number is expected to increase to almost 200 million worldwide by 2020. Lutein is a carotenoid currently used as a dietary supplement taken to treat and prevent the onset of AMD. The production of lutein is currently done through the cultivation of marigolds. Carotenoids are extracted from its petals, from which lutein is isolated.
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Our aim is to introduce a system capable of producing lutein from carotenoid precursors into a bacterial system. As these pathways are native to plants and nonexistent in bacteria, tne main challenge is obtaining every enzyme necessary to allow the pathway to occur. Strains of lycopene (a carotenoid precursor) producing bacteria already exist, and we expect to begin the synthesis from this point. Another main challenge is the regulation of genes required to proceed from lycopene to lutein. Lycopene is the precursor for a multitude of carotenoids, all of which are produced in pants due to necessity. To produce lutein, the lycopene must first undergo a reaction with the specific enzymes in order, ε-cyclase then β-cyclase. Having both enzymes in the system, though, allows reactions between β-cyclase and lycopene, which yields a product unable to be converted into lutein. Through regulatory measures such as altering gene expression levels, we plan to optimize the efficiency of the lutein synthesis pathway. We also aim to create a mathematical modeling system capable of correlating the expression levels of proteins to relevant efficiencies in similar synthesis pathways.
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We are also working on testing alternative plasmid maintenance systems that would minimize or eliminate the need for repeated antibiotic exposure in the field. Currently, iGEM uses chloramphenicol as a negative selection pressure to maintain plasmids within cells. While chloramphenicol or another antibiotic may be used initially as a selective agent to identify proper transformants, toxin/antitoxin (TA) systems are capable of maintaining plasmids during growth, removing the need to constantly replenish antibiotics in the growth media. The Hok/Sok system is a toxin/antitoxin system that involves a quick degrading antitoxin (Sok) and a long-lasting toxin (Hok). Cells with the Hok/Sok plasmid will produce large amounts of quick-degrading anti-toxin to inhibit the toxin, while those that eject the plasmid would not be able to produce Sok, thereby leading to cell death as Hok would still be present within the bacterium.  
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Antibiotic resistence is a necessary selection factor for transgenic bacteria using plasmids as vectors. This staple of genetic engineering has been met with opposition with valid claims that the addition of antibiotics to the environment harms native species and poses a risk to unwanted antibiotic resistence through lateral gene transfer.
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The Hok/Sok system has naturally evolved in bacteria as a means of plasmid retention, and is capable of addressing the issue by providing a selection factor for plasmid retention without the dangers of antibiotics and risk of lateral gene transfer. The Hok (host killing) gene codes for a mRNA which lies dormant in its initial secondary structure. As it is degraded by exonuclease, it assumes a translatable secondary structure which produces an apoptosis triggering protein. The Sok (suppression of killing) gene codes for a mRNA transcript that binds to the Hok mRNA, preventing it from being translated. The complex is eventually degraded by nuclease. Hok has a half life of 20 minutes, while Sok has a half life of 30 seconds. As long as both genes are present, the cell remains alive. After cell division, should the cell not retain the plasmid of interest which contains Hok/Sok, Hok mRNA remains the cytoplasm for 20 minutes, while remaining Sok is degraded. Since the cell does not contain a Sok gene, no Sok is being produced to save the cell from being killed by Hok. This system is very similar to current antibiotic resistence systems, only without the necessecity for antibiotics themselves, resolving the issue of encironmentally safe plasmid retention.  
  
 
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Revision as of 15:50, 15 July 2015