|Title||Rapid synthesis and screening of chemically activated transcription factors with GFP-based reporters.|
|Publication Type||Journal Article|
|Year of Publication||2013|
|Authors||R McIsaac, S, Oakes, BL, Botstein, D, Noyes, MB|
|Journal||J Vis Exp|
|Keywords||Base Sequence, Connexin 43, Flow Cytometry, Green Fluorescent Proteins, Humans, Molecular Sequence Data, Peptide Fragments, Plasmids, Protein Engineering, Synthetic Biology, Transcription Factors, Yeasts|
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 ligand-binding 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.
|Alternate Journal||J Vis Exp|