Team:uOttawa

This year, the uOttawa iGEM team is developing a library of toggle switches in Saccharomyces cerevisiae. Our toggle switch is a genetic network consisting of two halves: a tet-repressible promoter driving expression of the lac repressor, and a lac-repressible promoter driving expression of the tet repressor. Each half of the toggle switch represses the other, which creates a bimodal genetic network that switches at a specific threshold. By generating a library of different tet-repressible and lac-repressible promoter biobricks, we can create a set of toggle switches with varied dynamics. We are creating our library of promoters by biobricking an existing proximal promoter library (Ellis et al., 2009) and combining the proximal promoters with various distal promoter biobricks. These promoter biobricks will greatly expand the existing yeast biobrick collection in the Registry of Standard Biological Parts. We will be attempting two parallel methods of combining each half of the toggle switch. The first method involves cloning each half into a yeast strain of a particular mating type, and then mating the two strains together to generate the toggle switch in a diploid. The second method entails cloning each half into a recombination targeting vector of our own design, and assembling both halves into a single haploid strain via PCR-mediated homologous recombination. This recombination method would be immensely useful in generating any genetic network involving multiple transcriptional units; each unit would be cloned into our targeting vectors, would be amplified via PCR using standard primers, and could then be recombinated into a single locus in yeast. The team also aims to create a dual-colour toggle switch by tagging each repressor with a different fluorescent protein. This involves identifying the best two fluorescent proteins to use simultaneously for analysis by flow cytometry. The dual-colour toggle switch allows the dynamics of the switch to be studied more directly, rather than inferring the dynamics with the use of an intermediate such as anhydrous tetracycline. Each component of our project, including biobricked promoters, tet and lac repressors, and fluorescent proteins, as well as our targeting vectors and testing plasmids, will be submitted to the Registry of Standard Biological Parts. This project will therefore contribute valuable yeast protocols and yeast biobricks to the Registry and to other iGEM teams.