Part:BBa_K4896969
SRE cholesterol responsive promoter
Our project involves the use of engineered promoter/transcription factor pairs coupled with RNA regulatory elements to manipulate cellular processes of practical importance. The test system chosen is a synthetic cholesterol regulation circuit designed to downregulate cholesterol biosynthesis in response to elevated cholesterol levels. This is a two-step circuit that will both detect and inhibit cholesterol levels in liver cells. HEPG2 cells will be modified with the plasmid to detect changes in cholesterol levels and inhibit cholesterol biosynthesis at high levels. High cholesterol levels can have devastating consequences including atherosclerosis, stroke, and kidney disease. Further development of the SCRC circuit would help to treat the underlying cause of some of the most deadly conditions in the United States. The project has two thrusts: developing the sensing elements and developing the inhibitory element. For the sensing element, we have created a cholesterol biosensor via an engineered promoter, pSRE. Cholesterol biosynthesis is regulated by the SREBP system that contains SRE elements. We moved several SRE elements into a CMV promoter which will hopefully become regulated by this system; this is pSRE. To verify this we used a dual luciferase assay. While the dual luciferase assay did not provide significant results. It did, however, allow us to characterize the SRE promoter as sensitive to cholesterol. These preliminary results show that the promoter is active at low cholesterol levels and inactive at high cholesterol levels.
We would like to create a circuit that would integrate the pSRE biosensor and a siRNA inhibition mechanism, by placing the siRNA under the control of pSRE, with the hopes to inhibit cholesterol production at high cholesterol levels.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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