Yeast Experiments

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 is realized to driving 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. Induction was done 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.
medium with 1.5% raffinose, 1.5% glycerol, and 1% ethanol as carbon sources. Induction was done 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.
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LucAssay result :
LucAssay result :
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                [[Image:Luciferase1.jpg]]
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          [[Image:Luciferase1.jpg]]
2XORE promoter have a minimal basal activity without oleate and we can see a proportionally increment when increasing oleate concentrations
2XORE promoter have a minimal basal activity without oleate and we can see a proportionally increment when increasing oleate concentrations

Latest revision as of 18:08, 10 September 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 is realized to driving 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. Induction was done 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 :

          Luciferase1.jpg

2XORE promoter have a minimal basal activity without oleate and we can see a proportionally increment when increasing oleate concentrations