Synthetic Cholesterol Regulatory Circuit
Synthetic Cholesterol Regulatory Circuit
Cholesterol is a fatty substance found in the cells of our body and is essential for various physiological functions, including the formation of cell membranes and the production of hormones. However, high levels of cholesterol in the bloodstream can be detrimental to our health. When there is an excess of cholesterol, it can build up in the walls of arteries, forming plaques that narrow and harden the arteries, a condition known as atherosclerosis. 39% of adults suffer from high cholesterol.
Synthetic biology seeks to apply engineering and design principles to living systems, to design them to achieve specific ends. This can be applied to any living system and has broad implications in any sector involving life e.g. the environment, sustainability, or health. Our project seeks to apply synthetic biology to a significant problem of societal relevance. The specific example chosen is hypercholesterolemia, within the human health sector, since this is a very large problem with an imperfect solution.
According to the CDC, two in five adults in the United States suffer from high cholesterol. High levels of cholesterol increase one's risk of developing heart disease and stroke, which are the leading causes of death in Americans. The most common treatment for high cholesterol is statins. However, statins not only have a myriad of side effects, but they also work in varying capacities in everyone, which affects outcomes. An approach based on synthetic biology that would regulate the synthesis of cholesterol using a synthetic cholesterol regulatory circuit could provide an alternative. We have named this circuit the Decholesterolizer. We are developing the Decholesterolizer in a two-step circuit that will first detect cholesterol levels and then inhibit cholesterol production. Since the liver is the primary organ that is involved in the synthesis of cholesterol, we have selected to perform our studies in human liver cells. The liver HepG2 cells were modified with a plasmid to detect changes in cholesterol levels and in parallel, with a siRNA to inhibit cholesterol biosynthesis. Cholesterol biosynthesis is regulated by the sterol regulatory element binding proteins (SREBP) system which contains sterol regulatory elements (SRE). These SRE elements were transferred into a cytomegalovirus (CMV) promoter creating an engineered pSRE promoter that acts as a cholesterol biosensor. Verification of the pSRE promoter function was done using a dual luciferase assay. The inhibitory part of the Decholesterolizer will contain a short hairpin RNA (shRNA) that will knock out squalene monooxygenase (SQLE) to down-regulate cholesterol production. The design of a vector containing the shRNA was successfully created. Looking forward, the detection and inhibition part of the Decholesterolizer will be transferred into the same vector. The shRNA unit would be under the control of the pSRE promoter, which would inhibit cholesterol production at high cholesterol levels.
Provided quantitative characterization data for our SRE promoter and siRNA-SQLE.
Will provide $3400 worth of IDT product including primers and siRNAs.
Will promote Illinois Tech iGEM team and project in blog post.
Provided $2500 to cover most of the Team Grand Jamboree Fee.