Difference between revisions of "Part:BBa K2247000"

Latest revision as of 10:02, 30 October 2017

pGalS promoter of S. cerevisiae

This is a modified version of the yeast endogenous Gal1 promoter, from which expression can be induced by galactose, or the yeast Gal4 activator. pGalS also contains the upstream activating sequence (UAS) which Gal4 can bind, and can serve as the promoter in a standard yeast-two-hybrid system.

Usage and Biology

This part is a Gal promoter from the yeast S. cerevisiae, modified from the endogenous Gal1 promoter. Endogenous Gal1 promoter contains upstream activating sequence (UAS), where lies the binding sites of the yeast Gal4 activator. Similar to pGal1, the expression from pGalS can be induced by Gal4 protein, or simply the addition of galactose, with a higher strength than pGal1. This promoter can serve as an positively regulated promoter in the S. cerevisiae system, or drive the expression of the reporter gene in any standard version of yeast-two-hybrid system. In our system, this promoter coupled the sensor and the report part, driving the expression of reporter genes when Gal4 AD and BD domains, each fused with an anti-aflatoxin B1 antibody, associated in the presence of aflatoxin B1.

Characterization

Figure 1. Yeast Hexose Transporter 2 (HXT2) expression under the pGalS promoter upon Gal4 induction.

This figure shows the Gal4-inducible expression from the pGalS promoter (BBa_K2247000). The yeast strain JDYY001, of which all endogenous HXT-related genes and Gal4 and Gal80 are knocked out, was transformed with the pGalS-HXT2 plasmid with either a plasmid encoding Gal4 (experimental group) or an empty vector (control group), and plated on media where glucose was the sole carbon source. As all hexose transporters were knocked out, this strain of yeast was able to grow on the glucose-only media only when HXT2 could be expressed from the pGalS promoter. Therefore, the growth of JDYY001 on glucose-only media was chosen as the sign of gene expression under pGalS. Upper half, yeast cells transformed with pGalS-HXT2 and Gal4 plasmids. Lower half, control cells transformed with pGalS-HXT2 and empty vector. The growth of the experimental but not the control group showed that expression can be potently induced by Gal4 from pGalS promoter, as expected.

Figure 2. Yeast GFP expression under the pGalS promoter upon Gal4 induction.

This figure shows another proof of the Gal4-inducible expression from the pGalS promoter (BBa_K2247000). The above mentioned yeast strain JDYY001 was transformed with plasmids encoding pGalS-GFP and Gal4. These representative images showed successful expression of GFP under pGalS induced by Gal4 protein, also validating the function of pGalS promoter.

Sequence and Features