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Galactose dose response 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 previous dose response experiment using the fluorometer (will link the exp) did not show a steady increase of GFP expression with increasing galactose concentrations. Therefore the experiment was repeated using lower concentrations of this inducing agent. We have therefore tested media containing: 0.5%, 0.1%, 0.2%, 0.3%, 0.5%, 1% and 2% of galactose.
Protocol
1. Yeast 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
FACS data
The graph above summarises the FACS data, and shows that the intensity of GFP expressing cells increases in response to the percentage of galactose in the growth medium. The GAL1 promoter in our construct showed a high degree of sensitiveity to the inducing agent, with concentraitons as low as 0.01% having significant inducing potential.
Conclusion
The experiment clearly showed that the percentage of cells expressing GFP was exquisitely sensitive to the presence of galactose, with the dose response saturating above 0.5% galactose. This therefore clearly shows that the GAL1 promoter is highly sensitive, but that as a synthetic biology part, it may not exhibit ideal linear responses to inducing agent for some applications. The observed GFP induction curve suggests that the promoter behaves as an analogue switch only over a very narrow range of galactose concentrations.
