Luciferase assay

We have cloned one(1ORE)and two tandem copies of the oleate response elements (2XORE) from the FOX3 gene inserted upstream of a minimal CYC1 promoter.This promoter drives the expression of firefly luciferase.
For luciferase assays and the preparation of protein extracts, transformants were grown in rich
medium with 1.5% raffinose, 1.5% glycerol, and 1% ethanol as carbon sources. Cell were induced with different concentrations of oleate overnight.Cells were lysed in 100 mM potassium phosphate pH 7.8, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol using glass beads (diameter 0.45 mm). Cell debris were removed by centrifugation at 15 000 x g at 4°C for 20 min.Luciferase activities are expressed in relative light units/pg protein and protein concentrations were determined by the method of Bradford using bovine serum albumin as a standard.

LucAssay result :

           [[Image:luc.jpg]]

2XORE promoter has a minimal basal activity without oleate and we can see a proportional increment when increasing oleate concentrations.