title: Construction of a cytosolic firefly luciferase reporter cassette for use in PCR-mediated gene deletion and fusion in Saccharomyces cerevisiae.
abstract: Monitoring promoter response to environmental changes using reporter systems has provided invaluable information regarding cellular state. With the development of in vivo luciferase reporter systems, inexpensive, sensitive and accurate promoter assays have been developed without the variability reported between in vitro samplings. Current luciferase reporter systems, however, are largely inflexible to modifications to the promoter of interest. To overcome problems in flexibility and stability of these expression vectors, we report the creation of a novel vector system which introduces a cytosol-localized Photinus pyralis luciferase [LUC*(-SKL)] capable of one-step, in vivo measurements into a promoter-reporter system via PCR-based gene deletion and fusion. After introduction of the reporter under HUG1 promoter control, cytosolic localization was confirmed by fluorescence microscopy. The dose-response of this novel construct was then compared with that of a similar HUG1Δ::yEGFP1 promoter-reporter system and shown to give a similar response pattern.
authors.affiliation: Cain Department of Chemical Engineering, Louisiana State University, Baton Rouge, LA 70803, USA.
authors.forename: W B
authors.initials: WB
authors.lastname: Ainsworth
authors.forename: C M
authors.initials: CM
authors.lastname: Rome
authors.forename: M A
authors.initials: MA
authors.lastname: Hjortsø
authors.forename: M G
authors.initials: MG
authors.lastname: Benton
mesh_terms: D000818:Animals; D003600:Cytosol / Q000201:enzymology; D004790:Enzyme Induction; D048888:Fireflies / Q000201:enzymology / Q000235:genetics; D017353:Gene Deletion; D050939:Gene Fusion; D055786:Gene Knockout Techniques; D017930:Genes, Reporter; D005818:Genetic Engineering; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D049409:Luciferases, Firefly / Q000235:genetics* / Q000378:metabolism; D049408:Luminescent Agents / Q000378:metabolism; D008163:Luminescent Measurements; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics
chemical_list: C403293:HUG1 protein, S cerevisiae; D049408:Luminescent Agents; D020648:Peptide Elongation Factor 1; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C410826:TEF1 protein, S cerevisiae; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D049409:Luciferases, Firefly