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Optimization of the Tet-On system in Saccharomyces cerevisiae


Synthetic biology in Saccharomyces cerevisiae or budding yeast has proved indispensable in metabolic engineering, gene circuit engineering, and molecular and cell biology. These applications require robust “plug-and-play” inducible promoter systems for tight and tunable control of gene expression, but current inducible systems have limitations. We have incorporated the parts of the Tet-On system into the MoClo YTK system. With these parts we have built and optimized the Tet-On system in S. cerevisiae through multiple design iterations. Through different combinations of the rtTA transactivators and constitutive promoters, we have created strains with varying responses to doxycycline induction that will be useful for different applications. These systems boast extremely tight control over basal activity indistinguishable from the background strain, nanomolar sensitivities to doxycycline, and maximum output surpassing the strong pTDH3 promoter. Not only are these systems well-characterized and ready-to-use but the incorporation of the parts into the MoClo YTK allows them to be easily customized for specific applications.

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