Parallel High‐Throughput Automated Assays to Measure Cell Growth and Beta‐Galactosidase Reporter Gene Expression in the Yeast Saccharomyces cerevisiae

Andrew D. Napper1, Nuzhat Motlekar2, Rogerio Alves de Almeida3, Graham D. Pavitt3

1 Nemours Center for Childhood Cancer Research, Wilmington, Delaware, 2 Penn Center for Molecular Discovery, Institute for Medicine and Engineering, and Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, Pennsylvania, 3 Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch100119
Online Posting Date:  January, 2011
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Abstract

Parallel high‐throughput automated assays are described for the measurement of cell growth and β‐galactosidase reporter gene expression from a single culture of the yeast S. cerevisiae. The dual assay measures the effect of test compounds on expression of a specific gene of interest linked to the β‐galactosidase reporter gene, and simultaneously tests for compound toxicity and other effects on cell growth. Examples of assay development and validation results are used to illustrate how this protocol may be used to screen two yeast cell lines in parallel. Yeast cells are grown overnight in V‐bottom polypropylene 384‐well plates, after which portions of the cell suspension are transferred to clear and to white flat‐bottom 384‐well plates for measurement of cell growth and reporter gene expression, respectively. Cell growth is determined by measurement of absorbance at 595 nm, and β‐galactosidase expression is quantified by Beta‐Glo, a commercially available luminescent β‐galactosidase substrate. Curr. Protoc. Chem. Biol. 3:1‐14 © 2011 by John Wiley & Sons, Inc.

Keywords: cell growth; yeast; reporter gene; luciferase; cell‐based assay; model organism; mutant gene; β‐galactosidase; luminescence

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Frozen Yeast Stocks, Optimization of Cell Growth Conditions, and Determination of the Effect of DMSO on Yeast Cell Growth
  • Basic Protocol 2: Yeast End‐Point Growth Assay: Absorbance at 595 nm
  • Basic Protocol 3: β‐Galactosidase Reporter Expression Time Course in Yeast
  • Basic Protocol 4: Luminescence End‐Point β‐Galactosidase Expression Assay
  • Basic Protocol 5: Single‐Concentration Compound Screening
  • Basic Protocol 6: Data Analysis and Hit Selection
  • Basic Protocol 7: Dose‐Response Testing, Curve Fitting, IC50 Determination, and Hit Confirmation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Frozen Yeast Stocks, Optimization of Cell Growth Conditions, and Determination of the Effect of DMSO on Yeast Cell Growth

  Materials
  • Matched pair of yeast strains
  • YPD plates (contain YPD medium with an addition of 20 g/liter agar; supplied by Formedium)
  • YPD medium (10 g/liter yeast extract, 20 g/liter bacto peptone, and 20 g/liter glucose; available from Sigma)
  • Freezing medium: YPD medium with 15% (v/v) glycerol
  • Dry ice
  • 0% to 1.6% (v/v) final dimethyl sulfoxide (DMSO)
  • 30°C incubator
  • 250‐ml Erlenmeyer flasks
  • Shaking incubator
  • Freezer vials for yeast stocks: 0.2‐ml snap‐cap PCR tubes (from any supplier of standard laboratory accessories)
  • Polypropylene V‐bottom 384‐well plates (Greiner Bio‐One, cat. no. 781280)
  • Pipetting workstation equipped with 384‐tip pipetting head (e.g., JANUS from Perkin Elmer or equivalent; Rudnicki and Johnston, )
  • Reagent dispenser (e.g., Multidrop‐384 reagent dispenser from Thermo Scientific; Rudnicki and Johnston, )
  • Plate reader capable of reading absorbance and luminescence in 384‐well plates (e.g., EnVision multimode plate reader from Perkin Elmer)
  • Clear 384‐well plates (Corning, cat. no. 3702)
  • Breathe‐Easy sealing films (Sigma, cat. no. Z380059)

Basic Protocol 2: Yeast End‐Point Growth Assay: Absorbance at 595 nm

  Materials
  • YPD medium (10 g/liter yeast extract, 20 g/liter bacto peptone, and 20 g/liter glucose; available from Sigma)
  • Test compounds in DMSO
  • Yeast strains, wild type and mutant
  • Z buffer (see recipe)
  • Polypropylene V‐bottom 384‐well plates (Greiner Bio‐One, cat. no. 781280)
  • Pipetting workstation equipped with pintool consisting of 384 pins with nominal transfer volume of 100 nl (e.g., JANUS MDT from Perkin Elmer or equivalent; Rudnicki and Johnston, )
  • Pipetting workstation equipped with 384‐tip pipetting head (e.g., JANUS from Perkin Elmer or equivalent; Rudnicki and Johnston, )
  • Vortex
  • Breathe‐Easy sealing films (Sigma, cat. no. Z380059)
  • 30°C incubator
  • White 384‐well plates (Corning Life Sciences, cat. no. 3652)
  • Clear 384‐well plates (Corning Life Sciences, cat. no. 3702)
  • Plate reader capable of reading absorbance and luminescence in 384‐well plates (e.g., EnVision multimode plate reader from Perkin Elmer)

Basic Protocol 3: β‐Galactosidase Reporter Expression Time Course in Yeast

  Materials
  • Yeast cell suspension prepared in protocol 2, step 6
  • Z buffer
  • Beta‐Glo assay system (Promega, cat. no. E4780)
  • White, flat‐bottomed 384‐well plates
  • Reagent dispenser (e.g., Multidrop‐384 reagent dispenser from Thermo Scientific; Rudnicki and Johnston, )
  • Pipetting workstation equipped with 384‐tip pipetting head (e.g., JANUS from Perkin Elmer or equivalent; Rudnicki and Johnston, )
  • Plate reader capable of reading absorbance and luminescence in 384‐well plates (e.g., EnVision multimode plate reader from Perkin Elmer)

Basic Protocol 4: Luminescence End‐Point β‐Galactosidase Expression Assay

  Materials
  • Compound stocks (10 mM in DMSO)
  • Dimethyl sulfoxide (DMSO)
  • 384‐well V‐bottom polypropylene plates
  • Reagent dispenser (e.g., Multidrop‐384 reagent dispenser from Thermo Scientific; Rudnicki and Johnston, )
  • Pipetting workstation equipped with pintool consisting of 384 pins with nominal transfer volume of 100 nl (e.g., JANUS MDT from Perkin Elmer or equivalent; Rudnicki and Johnston, )
  • Microsoft Excel: for calculation of percent inhibition or activation (if screening database is not available)
  • GraphPad Prism or equivalent: for graphing data and curve fitting for IC 50 and EC 50 calculation (if screening database is not available)
  • OpenHTS (CeuticalSoft), ActivityBase (IDBS), or equivalent screening database: for calculation of percent inhibition or activation, comparison of data sets and selection of hits
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Figures

Videos

Literature Cited

Literature Cited
   Alves de Almeida, R., Burgess, D., Shema, R., Motlekar, N., Napper, A.D., Diamond, S.L., and Pavitt, G.D. 2008. A Saccharomyces cerevisiae cell‐based quantitative beta‐galactosidase assay compatible with robotic handling and high‐throughput screening. Yeast 25:71‐76.
  Inglese, J., Johnson, R.L., Simeonov, A., Xia, M., Zheng, W., Austin, C.P., and Auld, D.S. 2007. High‐throughput screening assays for the identification of chemical probes. Nat. Chem. Biol. 3:466‐479.
   Lederberg, J. 1950. The beta‐D‐galactosidase of Escherichia coli, strain K‐12. J. Bacteriol. 60:381‐392.
   Miller, J.H. 1972. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
   Motlekar, N., de Almeida, R.A., Pavitt, G.D., Diamond, S.L., and Napper, A.D. 2009. Discovery of chemical modulators of a conserved translational control pathway by parallel screening in yeast. Assay Drug Dev. Technol. 7:479‐494.
   Murata, Y., Watanabe, T., Sato, M., Momose, Y., Nakahara, T., Oka, S., and Iwahashi, H. 2003. Dimethyl sulfoxide exposure facilitates phospholipid biosynthesis and cellular membrane proliferation in yeast cells. J. Biol. Chem. 278:33185‐33193.
   Rudnicki, S. and Johnston, S. 2009. Overview of liquid handling Instrumentation for high‐throughput screening applications. Curr. Protoc. Chem. Biol. 1:43‐54.
   Sambrook, J., and Russell, D.W. 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
   Warringer, J. and Blomberg, A. 2003. Automated screening in environmental arrays allows analysis of quantitative phenotypic profiles in Saccharomyces cerevisiae. Yeast 20:53‐67.
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