Measurement of Induction of the GAL1 Promoter Over Time in Construct GAL1p-(Npep-GFP)

Aim

The aim of this experiment was to test the response of the GAL1 promoter in the presence of galactose over time, by measuring the expression of GFP, the downstream gene. Construct Gal1p-(Npep-GFP) was used in the experiments described here

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

flow cytometry data showing the changes to the GFP expression (peak to right) and non GFP expressing cells (peak to left) over time as a result of galactose being added. There are two significant peaks. The peak to the left of the graph represents the number of cells which did not express GFP and the peak to the right the number of the cells which did express GFP. The highest peak to the left is produced by the cells before adding galactose and the peak to the right is not present thus there is no GFP expression by the cells at time zero of the experiment, hence no natural GFP expression by the cells. As time increases there is an increased number of cells which are expressing GFP in the presence of galactose inducer, shown by the gradual increase of the peak to the right over time. The visible peak starts appearing 60 mins after adding galactose (the light blue line).

The graph above summarises the flow cytometry data, and shows that the intensity of GFP expressing cells increases over time after galactose has been added. The graph does not reach a plateau opver the time of the experiment. This may suggest that the cells have not expressed to their maximum capacity. Therefore to conclude, further experiments may be repeated with an increased galactose concentration or an increased period of time over which the experiment was carried out.

Conclusion

The experiment clearly showed that the percentage of cells expressing GFP increased to 68% after 167 minutes from the time that 0.1 % galactose was added to the culture medium. This therefore clearly showing that galactose has successfully induced the expression of the GFP from the GAL1-(Npep-GFP) construct. Expression induction was almost linear over this time, and after 167 minutes, the expression induction had still not reached a plateau.

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