uOttawa IGEM2009

uOttawa iGEM2009


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BBa_K234095 Characterization


We have contributed to the iGEM biobrick registry by submitting several parts which can be used in eukaryotic organisms, namely in Yeast and Mammalian cells. The Gal 1/10 divergent promoter is essentially composed of two promoters and is capable of driving gene expression bidirectionally. Gene expression is driven in one direction by the Gal 1 promoter and by the Gal 10 promoter in the other direction, both promoters are inducible by galactose present in the cell medium. The construct was isolated from the pRS4T123 plasmid, generously donated to us by the James Collins Lab. Due to it's divergent design, the dual promoter is compact and thus easy to work with. Hilary Phenix and Vida Adebi helped us with flow cytometry and they also generated a yeast strain containing a Gal 1/10 divergent promoter, with Gal 10 driving GFP expression. We characterized the Gal 10 component of the promoter in yeast by performing a dose response experiment where we induced the yeast with various concentrations of galactose and measured the resulting GFP output using a flow cytometer. We also contributed a CYC1 terminator containing a stop codon to the registry, a commonly used terminator sequence in yeast which was also isolated form the pRS4T123 plasmid . Including a stop codon with the terminator allows this construct to be fused with proteins in the biofusion format (assembly standard 23). We also submitted a CMV promoter, naturally found in the Cytomegalovirus. The submitted promoter was isolated from a plasmid that was created in our lab, pCMV is a constitutive promoter commonly used in mammalian cloning vectors. We submitted these parts in a effort provide at least some basic components to the registry that can be used by future iGEM teams who wish to engineer eukaryotic cells