NDLI: Construction of an engineered yeast with glucose-inducible emission of green fluorescence from the cell surface

Content Provider	Springer Nature Link
Author	Ye, K. Shibasaki, S. Ueda, M. Murai, T. Kamasawa, N. Osumi, M. Shimizu, K. Tanaka, A.
Copyright Year	2000
Abstract	An engineered yeast with emission of fluorescence from the cell surface was constructed. Cell surface engineering was applied to display a visible reporter molecule, green fluorescent protein (GFP). A glucose-inducible promoter GAPDH as a model promoter was selected to control the expression of the reporter gene in response to environmental changes. The GFP gene was fused with the gene encoding the C-terminal half of α-agglutinin of Saccharomyces cerevisiae having a glycosylphosphatidylinositol anchor attachment signal sequence. A secretion signal sequence of the fungal glucoamylase precursor protein was connected to the N-terminal of GFP. This designed gene was integrated into the TRP1 locus of the chromosome of S. cerevisiae with homologous recombination. Fluorescence microscopy demonstrated that the transformant cells emitted green fluorescence derived from functionally expressed GFP involved in the fusion molecule. The surface display of GFP was further verified by immunofluorescence labeling with a polyclonal antibody (raised in rabbits) against GFP as the first antibody and Rhodamine Red-X-conjugated goat anti-rabbit IgG as the second antibody which cannot penetrate into the cell membrane. The display of GFP on the cell surface was confirmed using a confocal laser scanning microscope and by measuring fluorescence in each cell fraction obtained after the subcellular fractionation. As GFP was proved to be displayed as an active form on the cell surface, selection of promoters will endow yeast cells with abilities to respond to changes in environmental conditions, including nutrient concentrations in the media, through the emission of fluorescence.
Starting Page	90
Ending Page	96
Page Count	7
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	54
Issue Number	1
e-ISSN	14320614
Language	English
Publisher	Springer-Verlag
Publisher Date	2000-07-14
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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