Timed Induction of Gal1 Promoter in pRS415

From 2010.igem.org

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<h1>Measurement of induction of the GAL1 promoter over time in construct GAL1p-(Npep-GFP)</h1>
 
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<h3>Aim</h3>
 
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<p>The aim of this experiment
 
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To test the response of the GAL1 promoter in the presence of galactose over time, by measuring the expression of GFP, the downstream gene.
 
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</p>
 
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<h3>Protocol</h3>
 
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<p>
 
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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. <br><br>
 
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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. <br>
 
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<br>
 
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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.
 
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<br><br>
 
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<h3>Results</h3>
 
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<br>
 
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<p>
 
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Text
 
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</p>
 
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<center>
 
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https://static.igem.org/mediawiki/2010/0/04/Gal-FACS.jpg
 
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</center>
 
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<h3>Conclusion</h3>
 
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<p>
 
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Text
 
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</p>
 
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</html>
 
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<br>
 

Latest revision as of 14:05, 25 October 2010