Luciferase assay
From 2007.igem.org
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| - | 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 | + | 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 | 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. | + | 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 : | ||
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[[Image:luc.jpg]] | [[Image:luc.jpg]] | ||
| - | 2XORE promoter | + | 2XORE promoter has a minimal basal activity without oleate and we can see a proportional increment when increasing oleate concentrations. |
Revision as of 16:41, 25 October 2007
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 :
2XORE promoter has a minimal basal activity without oleate and we can see a proportional increment when increasing oleate concentrations.
