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Timed Induction of Gal1 Promoter in pRS415
From 2010.igem.org
Contents |
Measurement of induction of the GAL1 promoter over time in construct GAL1p-(Npep-GFP)
Aim
The aim of this experiment To test the response of the GAL1 promoter in the presence of galactose over time, by measuring the expression of GFP, the downstream gene.
Protocol
1. Yeasts transformed with a plasmid carrying the GAL1p-(Npep-GFP) construct was inoculated overnight into 5 ml of synthetic defined (SD) medium with amino acids: his (0.2 %), met (0.2 %), ura (0.2 %), trp (0.2 %) and Raffinose (2 %) as the carbon source.
2. The following evening 861 µl of this cell culture were sub-cultured into a flask containing pre-warmed SD medium (50 mls) to achieve an optical density at 600nm of 0.3 by 10am the following morning.
3. At OD 600 of 0.30, a 1 ml sample was taken to represent the t=0 min sample, and then galactose addded to a final concentration of 0.1 % w/v to begin the promoter induction process. Samples were then taken every 20 minutes thereafter for a period of 170 minutes. All samples were pelleted (13000 rpm, 5min, 4 degrees C), washed once with PBS buffer and stored on ice. Once collected all samples were then dispensed in PBS and diluted by a factor of 1/20 for flow cytometry analysis.
Results
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Conclusion
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