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Generating Chromosome-Located Transcriptional Fusions to Fluorescent Proteins for Single-Cell Gene Expression Analysis in Pseudomonas syringae

  • José S. Rufián
  • Diego López-Márquez
  • Nieves López-Pagán
  • Murray Grant
  • Javier Ruiz-Albert
  • Carmen R. BeuzónEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1734)

Abstract

The last decade has seen significant effort directed toward the role of phenotypic heterogeneity in bacterial adaptation. Phenotypic heterogeneity usually refers to phenotypic diversity that takes place through nongenetic means, independently of environmental induced variation. Recent findings are changing how microbiologists analyze bacterial behavior, with a shift from traditional assays averaging large populations to single-cell analysis focusing on bacterial individual behavior. Fluorescence-based methods are often used to analyze single-cell gene expression by flow cytometry, fluorescence microscopy and/or microfluidics. Moreover, fluorescence reporters can also be used to establish where and when are the genes of interest expressed. In this chapter, we use the model bacterial plant pathogen Pseudomonas syringae to illustrate a method to generate chromosome-located transcriptional gene fusions to fluorescent reporter genes, without affecting the function of the gene of interest.

Key words

Phenotypic heterogeneity Gene expression Fluorescent reporter genes Single-cell methods Fluorescence microscopy Nongenetic variation Allelic Exchange 

Notes

Acknowledgments

The work was supported by a Project Grant from Ministerio de Economía y Competitividad (Spain) (BIO2015-64391-R) awarded to C.R. Beuzón and J. Ruiz-Albert. M.G. and J.R. acknowledge support from the BBSRC/EPSRC Synthetic Biology Research Centre (BB/M017982/1) funded under the UK Research Councils’ Synthetic Biology for Growth Programme. The work was cofunded by Fondos Europeos de Desarrollo Regional (FEDER).

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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • José S. Rufián
    • 1
  • Diego López-Márquez
    • 1
  • Nieves López-Pagán
    • 1
  • Murray Grant
    • 2
  • Javier Ruiz-Albert
    • 1
  • Carmen R. Beuzón
    • 1
    Email author
  1. 1.Dpto. Biología Celular, Genética y Fisiología, Instituto de Hortofruticultura Subtropical y MediterráneaUniversidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC)MálagaSpain
  2. 2.School of Life SciencesUniversity of WarwickConventryUK

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