New Basic Part
The cholesterol biosensor (BBa_K4896969) is based on a viral genetic element, the cytomegalovirus promoter, that was engineered to add 7 slightly modified human sterol regulatory elements, SREs, from the cholesterol biosynthetic pathway genes. This created a new engineered promoter, pSRE, that is responsive to cholesterol level, and is the cholesterol sensor in our overall circuit. We have validated the function of this promoter in using a luciferase reporter system.
During cholesterol biosynthesis, sterol regulatory element binding protein binds with the cleavage activating protein to form the SREBP-Scap unit. When cholesterol levels are high the SREBP-Scap unit binds to the Insig protein. This allows the unit to remain in the membrane of the endoplasmic reticulum. However, when cholesterol levels decrease, the SREBP-Scap unit unbinds from the Insig protein. Interactions with the Sec23 and Sec24 proteins then allow the unit to be transported to the Golgi apparatus by a COPII vesicle. Here the SREBP-Scap unit can interact with the Site 1 protease to perform the first cleavage event. The Site 2 protease then mediates the second cleavage event, leaving the nuclear form of the sterol regulatory element binding protein. This enters the nucleus, where it binds with the pSRE promoter. The pSRE promoter can now activate target genes essential for cholesterol biosynthesis.
This pSRE promoter was used to engineer the CMV promoter to create the cholesterol biosensor essential for our hero’s work.