Team:uOttawa/Biobricks2

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Gal 1/10 divergent promoter

Gal 1/10 divergent promoter

Contents

Usage and Biology

The Gal 1/10 divergent promoter contains the Gal10 promoter driving expression to the left and the Gal1 promoter driving expression to the right. Both these promoters are repressed by the presence of glucose in the cell medium and activated by the presence of galactose. This part was isolated from the pRS4D1 plasmid generously donated to us by Dr. Jim Collins[1].

Characterization

Hilary Phenix and Vida Adebi, members of our lab, both introduced us to flow cytometry and generated a BY4742 yeast strain containing the Gal 1/10 promoter with Gal10 driving GFP expression. We used this yeast strain to characterize the Gal 10 promoter by performing a dose response experiment. Yeast were grown overnight in YPD + 1% Raff + 1% Adenine. Then the cells were inoculated to a final OD of 0.02 in varying concentrations of galactose. After 5 hours, cell fluorescence was measured using a BeckmanCoulter FC500 MPL equipped with a 15-mW argon laser. GFP excitation was performed at 600v and detected using a 525±15 bandpass filter (FL1).

Results:

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Figure 1. Dose response curve of GFP expression in response to varying concentrations of galactose. The pink line represents wild type BY4742 yeast cells, and the green line represents BY4742 cells containing our Gal1/10 GFP construct.

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Figure 2. Flow cytometry graphic comparing GFP expression under the fully induced Gal10 promoter (from Gal1/10) to a minimally induced Gal10 promoter (from Gal1/10). The pink population highlights GFP expression at a concentration of 0.00064% galactose. The population in green illustrates GFP expression at a concentration of 0.4% galactose.

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Figure 3. Flow cytometry graphic showing medium induction of GFP under the Gal10 promoter (from Gal1/10). This measurement was taken after growing cells for 5 hours at a concentration of 0.016% galactose.

References:

[1] Murphy et al.(2006): Combinatorial promoter design for engineering noisy gene expression. PNAS,104:12726-12731