Engineering a two-gene system to operate as a highly sensitive biosensor or a sharp switch upon induction with β-estradiol

Abstract

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.

Laccase is a multitasking protein for synthetic gene circuits in the yeast Saccharomyces cerevisiae

“…Each of the other two systems demanded to assemble two new TUs. One TU was for TtLcc1 expression under the control of a synthetic regulated promoter, either 2xlex2Op_pCYC1core [ 25 ] or pCYC1min_tetOp [ 26 ] (see Fig. 3 A and S3B-D).…”

Hybrid Boolean gates show that Cas12c controls transcription activation effectively in the yeast S. cerevisiae

“…We tested Cas12c behavior as a signal carrier in synthetic gene hybrid gates—where translation was downregulated by up-to-two riboswitches responding to tetracycline ( Kötter et al, 2009 ). For a comparison, we implemented several different hybrid gates that made use of transcription factors based on either type V denAsCas12a ( Kleinstiver et al, 2019 ; Yu and Marchisio, 2021 ) or LexA-HBD (hER) (i.e., the bacterial protein LexA fused to the hormone-binding domain of the human estrogen receptor) ( Louvion et al, 1993 ; Zhou et al, 2022 ). Alternatively, we also employed promoters regulated by internal pathways (e.g., the GAL1 promoter).…”