Yeast Experiments
From 2007.igem.org
| (One intermediate revision not shown) | |||
| Line 1: | Line 1: | ||
| + | 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. | ||
| Line 4: | Line 5: | ||
LucAssay result : | LucAssay result : | ||
| - | + | [[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 :
2XORE promoter have a minimal basal activity without oleate and we can see a proportionally increment when increasing oleate concentrations
