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Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

Author(s): McIsaac, R Scott; Oakes, Benjamin L; Botstein, David; Noyes, Marcus B

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Abstract: Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligandbinding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.
Publication Date: 2013
Electronic Publication Date: 26-Nov-2013
Citation: McIsaac, R Scott, Oakes, Benjamin L, Botstein, David, Noyes, Marcus B. (2013). Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters. Journal of Visualized Experiments, 81), 10.3791/51153
DOI: doi:10.3791/51153
EISSN: 1940-087X
Pages: 1 - 12
Type of Material: Journal Article
Journal/Proceeding Title: Journal of Visualized Experiments
Version: Final published version. This is an open access article.

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