Version 1 (V1)
- pSRE: pCMV containing SRE elements
- Nluc: Nano luciferase under pSRE control for insert verification
- CMV promoter: constitutive promoter
- Neo: Neomycin resistance
- Ampicillin: Ampicillin resistance
The V1 vector contains our engineered SRE promoter and one luciferase gene.
The SRE promoter will act as a cholesterol biosensor. 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.
Luciferase is a reporter gene that will indicate if the promoter is on or off. One luciferase gene was added to V1 to validate the function of the SRE promoter.
The results from the assay were inconclusive. An improved validation method was added to V2.
Version 2 (V2)
- pSRE: pCMV containing SRE elements
- Luciferase: Firefly luciferase under pSRE control for insert verification
- CMV promoter: constitutive promoter
- Rluc: Renilla luciferase under pSCMV control for insert verification
- Neo: Neomycin resistance
- Ampicillin: Ampicillin resistance
The V2 vector contains our engineered SRE promoter and two luciferase genes.
The SRE promoter will act as a cholesterol biosensor. 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.
Luciferase is a reporter gene that will indicate if the promoter is on or off. Two luciferase genes were added to V2 to perform the dual luciferase assay to validate the function of the SRE promoter.
The results of the dual luciferase assay showed that the pSRE is sensitive to changes in cholesterol levels. The pSRE is active at low cholesterol levels and inactive at high levels.
Version 2.5 (V2.5)
The IITChicago team is working to develop vector V2.5. This version will contain a redesigned SRE promoter that will be activated at high cholesterol levels.
This is a validation vector that is being used by the regulatory response team to test different siRNA’s and their ability to inhibit cholesterol synthesis. The siRNA’s are targeting rate limiting steps in the cholesterol synthesis pathway. The team is quantifying how well each siRNA inhibits cholesterol synthesis to identify ideal candidate(s) to be included in the final vector.
Our initial circuit design was composed of a cholesterol sensor pSRE, and a response element, a siRNA that inhibited some gene in cholesterol biosynthesis. There are many genes that we are investigating; our team has so far validated an effective siRNA against SQLE, squalene epoxidase. In this fashion, the cell can sense cholesterol level and adjust cholesterol biosynthesis. However, when we began to validate the sensor, we realized that our circuit worked in the wrong direction, inhibiting cholesterol synthesis when cholesterol is low and allowing it to continue when cholesterol is high.
Version 3 (V3)
The IITChicago team is working to develop vector V3. This version will contain the pSRE cholesterol biosensor and siRNA cholesterol regulator.
In our most current design, we added an inverter element between the sensor element (pSRE) and the response element (the siRNA) to invert the regulation of siRNA.
- In a low cholesterol condition, pSRE will be activated causing repressor X to be transcribed, which will bind to pX (the promoter for the siRNA sequence) and prevent the siRNA from being transcribed, allowing cholesterol synthesis to proceed.
- In a high cholesterol condition, pSRE will not be active, stopping expression of X which then allows pX to turn on, producing the siRNA and inhibiting cholesterol production.
The X protein is engineered to contain a degron, which are tunable elements providing various degradation rates. This allows us to tune the degradation rate to eliminate X rapidly enough when pSRE is off, but allow it to accumulate when pSRE is on. We are currently investigating various X repression systems (e.g. TET, PIT) and degron elements.