The human estrogen receptor has been used for about thirty years, in the yeast S. cerevisiae, as a component of chimeric transcription factors. Its ligand, β-estradiol, permits to control the protein translocation into the nucleus and, as a consequence, the expression of the gene(s) targeted by the synthetic transcription factor. Activators that are orthogonal to the yeast genome have been realized by fusing the human estrogen receptor to an activation and a DNA-binding domain from bacteria, viruses, or higher eukaryotes. In this work, we optimized the working of a β-estradiol-sensing device—in terms of detection range and maximal output signal—where the human estrogen receptor is flanked by the bacterial protein LexA and either the strong VP64 (from herpes simplex virus) or the weaker B42 (from E. coli) activation domain. We enhanced the biosensor performance by thoroughly engineering both the chimeric activator and the reporter protein expression cassette. In particular, we constructed a synthetic promoter—where transcription is induced by the chimeric activators—based on the core sequence of the yeast CYC1 promoter, by tuning parameters such as the length of the 5′ UTR, the distance between adjacent LexA binding sites (operators), and the spacing between the whole operator region and the main promoter TATA box. We found a configuration that works both as a highly sensitive biosensor and a sharp switch depending on the concentration of the chimeric activator and the strength of its activation domain.
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