Luciferase assay
From 2007.igem.org
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| - | We | + | We 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 | + | For luciferase assays and preparation of protein extracts, transformants were grown in rich |
| - | medium with 1.5% raffinose, 1.5% glycerol | + | 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 : | LucAssay result : | ||
Revision as of 07:46, 26 October 2007
We 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 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 :
2XORE promoter has a minimal basal activity without oleate and we can see a proportional increment when increasing oleate concentrations.
