Gal 1/10

GAL 1- GAL 10 is a divergent promoter region of Saccharomyces cerevisiae, allowing it to regulate two genes simultaneously. Each complimentary strand promotes translation in opposing directions. Galactose induces transcription, while Glucose serves as a repressor. The promoter is inducible by as low as 2% Galactose the in medium.

It is particularly useful for measuring protein-protein interactions, expressing proteins with two subunits, or can simply be used to effectively cut the number of plasmids required for cloning in half, as two genes can be cloned into a single plasmid.
To validate this part we performed Real-Time PCR on yeast with our Leu-44a vector, containing two subunits of the same protein- 44a-Dioxygenase (44a). This enzyme contains to subunits: 44a Large Subunit (LS) and 44a Small Subunit (SS). We used Leu2 as a regulating gene, as it is found on the same plasmid, but its transcription is not dependent on the Gal 1/10 Promoter.

We grew 2 strains of yeast in SD media (containing glucose) for 36 hours. The strains were as follows:

1. 44a- Containing the vector shown in the map above, with the LS transcription being promoted by Gal 10 and SS transcription being promoted by Gal1.
2. CTRL strain, containing a similar but "empty" vector. This vector also contains the Leu2 gene, and Gal 1/10 promoters, but does not have the 44a genes inserted.

After growth, be split each strain into 2 treatments:

1. Induced: SG media, containing 2% galactose in the media.
2. Uninduced: SD media, containing 2% glucose in the media.
As the primers for the RT-PCR are gene specific to the subunits, we expected to see no product in the CTRL vectors, in both treatments. The results of this experiment are detailed in the graph below. After normalizing the raw expression data of our target genes to the expression of the Leu2 gene, we get the figures presented.

In the induced treatment, we see expression rise more than 20 fold over the uninduced treatment.
Additionally, we see approximately equal expression between the uninduced treatment and the control group, indicating the minor levels could be background noise caused by the non-specificity of primers, or primer-dimer amplification. (We used SYBR, and not a probe, therefore the possibility of such background noise arises)