title: A Saccharomyces cerevisiae cell-based quantitative beta-galactosidase assay compatible with robotic handling and high-throughput screening.
abstract: Reporter-gene assays that employ the Escherichia coli lacZ gene are ubiquitously employed in biological research. However, we were not able to readily identify a quantitative method that worked reliably with yeast (Saccharomyces cerevisiae) cells and that was compatible with high-throughput screening and robotic liquid handling tools. We have therefore adapted a commercially available assay employing a 6-O-beta-galactopyranosyl-luciferin substrate to provide the required sensitivity with minimal sample handling times. Our assay uses only one-tenth of the reagents suggested by the reagent manufacturer (Promega) for equivalent assays with mammalian cell cultures and produces rapid, sensitive and reproducible analysis with as little as 1 microl yeast cell culture and with < 100 cells. We demonstrate that the assay is compatible with yeast strains generated by the systematic yeast deletion project and functions equally well with genomically integrated or plasmid-encoded lacZ reporters and with cells grown in complex or defined media. The high-sensitivity, miniaturized format reduced sample handling required will make this assay useful for a wide range of applications.
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.
authors.forename: Rogerio Alves
authors.initials: RA
authors.lastname: de Almeida
authors.forename: Danielle
authors.initials: D
authors.lastname: Burgess
authors.forename: Reut
authors.initials: R
authors.lastname: Shema
authors.forename: Nuzhat
authors.initials: N
authors.lastname: Motlekar
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Napper
authors.forename: Scott L
authors.initials: SL
authors.lastname: Diamond
authors.forename: Graham D
authors.initials: GD
authors.lastname: Pavitt
mesh_terms: D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins / Q000235:genetics; D017930:Genes, Reporter; D007763:Lac Operon; D008156:Luciferases / Q000235:genetics; D008164:Luminescent Proteins / Q000032:analysis; D012371:Robotics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D001616:beta-Galactosidase / Q000032:analysis*
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C501391:GCN4 protein, S cerevisiae; D008164:Luminescent Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D008156:Luciferases; D001616:beta-Galactosidase