Positive Feedback Genetic Circuit Incorporating a Constitutively Active Mutant Gal3 into Yeast GAL Induction System.

The GAL expression system is the most frequently used induction technique in the yeast Saccharomyces cerevisiae. Here we report a simple but powerful genetic circuit for use with the GAL induction system. Briefly, an artificial positive feedback circuit was incorporated into the GAL regulatory network. We selected green fluorescent protein (GFP) as a reporter of GAL1 induction, and designed a strain that expressed a constitutively active Gal3 mutant protein (Gal3c) under control of the GAL10 promoter. In the resulting strain, GAL1 and GAL10 promoters regulate the expression of GFP and GAL3c, respectively. Because Gal3c sequesters the Gal80 repressor away from the Gal4 transcriptional activator in the same manner as the galactose-bound Gal3, the expressed Gal3c protein provokes further expression of GFP and Gal3c, yielding further enhancement of GAL induction. Thus, this GAL3c-mediated positive feedback circuit permits substantially enriched induction of a target gene at extremely low concentrations, or even in the absence, of galactose, while maintaining the strict glucose-mediated repression of the target.

Transplantation of the GAL regulon into G-protein signaling circuitry in yeast.

Here we present a successful transplantation of the GAL genetic regulatory circuitry into the G-protein signaling pathway in yeast. The GAL regulon represents a strictly regulated transcriptional mechanism that we have transplanted into yeast to create a highly robust induction system to assist the detection of on-off switching in G-protein signaling. In our system, we engineered yeast to drive the positive GAL regulatory gene in response to agonist-promoted G-protein signaling and to induce transcription of a green fluorescent protein (GFP) reporter gene under the control of the GAL structural gene promoter. Consequently, in response to agonist stimulation of G-protein-coupled receptors (GPCRs), the engineered yeast achieved more than a 150-fold increase in reporter intensity in up to 98% of cells, as determined by flow cytometric sorting. Surprisingly, agonist-stimulated induction of the GFP reporter gene was higher than that by galactose. Our approach to boost reporter gene induction could be applicable in establishing more efficient yeast-based flow cytometric screening systems for agonistic ligands for heterogeneous GPCRs.