Characterising the translational repression of GAL1p-[Npeptide-GFP] by trans expression of the MS2 protein

Aim

To test the effect of glucose on repression of the GAL1 promoter, and thus on shut-off of GFP expression from construct GAL1p-[Npeptide-GFP] construct over time.

Hypothesis

The presence of glucose should quickly repress the GAL1 promoter and therefore result in the overall reduction of the GFP intensity present within the cells; measurement of the rate of decay should identify the relative stability of the GFP protein

Protocol

1. Yeast transformed with the GAL1p-[Npeptide-GFP] construct were innoculated overnight in 5 mls of synthetic defined medium with amino acids; his (0.2 %), met (0.2%), ura (0.2%), trp (0.2 %) and Raffinose (2 %) as the carbon source. 2. Following overnight growth the cells were subcultured in fresh, pre-warmed SD medium (50 mls) containing galactose (a range of concentrations: see Results below) to obtain a predicted OD600 of 0.3 by 10 am the following morning. 3. The following morning, at an OD600 of 0.3, a sample (1 ml) was taken before and after the addition of glucose (2 %). Samples were then taken every 20 minutes thereafter for a period of 167 minutes. All samples were then pelleted (13000 rpm, 5 mins, 4 degreesC), washed once with PBS buffer and stored on ice. Once collected all samples were then dispenced in PBS and diuted by a factor of 1/20 for FACS analysis.

Results

Conclusion

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