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 Gal3csequesters the Gal80 repressor away from the Gal4 transcriptional activator in the same manner as the galactose-bound Gal3, the expressed Gal3cprotein 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.
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University , 1-1 Rokkodai, Nada, Kobe 657-8501, Japan.
Shintaro
S
Ryo
Graduate School of Science, Technology and Innovation, Kobe University , 1-1 Rokkodai, Nada, Kobe 657-8501, Japan.
Jun
J
Ishii
Graduate School of Science, Technology and Innovation, Kobe University , 1-1 Rokkodai, Nada, Kobe 657-8501, Japan.
Toshihide
T
Matsuno
Graduate School of Science, Technology and Innovation, Kobe University , 1-1 Rokkodai, Nada, Kobe 657-8501, Japan.
Yasuyuki
Y
Nakamura
Graduate School of Science, Technology and Innovation, Kobe University , 1-1 Rokkodai, Nada, Kobe 657-8501, Japan.
Daiki
D
Matsubara
Graduate School of Science, Technology and Innovation, Kobe University , 1-1 Rokkodai, Nada, Kobe 657-8501, Japan.
Masahiro
M
Tominaga
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University , 1-1 Rokkodai, Nada, Kobe 657-8501, Japan.|Graduate School of Science, Technology and Innovation, Kobe University , 1-1 Rokkodai, Nada, Kobe 657-8501, Japan.
Akihiko
0000-0003-1527-5288
A
Kondo
D003001:Cloning, Molecular; D025461:Feedback, Physiological; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005686:Galactokinase / Q000235:genetics* / Q000378:metabolism; D053263:Gene Regulatory Networks / Q000235:genetics; D005800:Genes, Fungal / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism
D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D005686:Galactokinase
GAL induction system; Gal3c; Saccharomyces cerevisiae; galactose; positive feedback circuit; protein production