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Promoter Fusions with Optical Outputs in Individual Cells and in Populations

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Pseudomonas Methods and Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1149))

Abstract

Reporter genes are widely used to quantify promoter activity, which controls production of mRNA through the interplay with RNA polymerases and transcription factors. Some of such reporters have either diffuse (lux) or focused (GFP) optical outputs that allow description of transcriptional activity in populations and in single cells. This chapter discusses the use of a dual reporter system GFP-luxCDABE that is placed in broad-host-range plasmids having origins of replication from RK2 and pBBR1. The value of this system is shown in Pseudomonas putida by characterizing the activity of the Pb promoter, which drives an operon for benzoate biodegradation in this bacterium. To this end we compare in the same cells bioluminescence as the output signal of the whole population and single cell-bound fluorescence caused by GFP expression and revealed by flow cytometry assays.

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References

  1. Browning DF, Busby SJ (2004) The regulation of bacterial transcription initiation. Nat Rev Microbiol 2:57–65

    Article  CAS  Google Scholar 

  2. Hermsen R, Tans S, ten Wolde PR (2006) Transcriptional regulation by competing transcription factor modules. PLoS Comput Biol 2:e164

    Article  Google Scholar 

  3. McAdams HH, Arkin A (1997) Stochastic mechanisms in gene expression. Proc Natl Acad Sci U S A 94:814–819

    Article  CAS  Google Scholar 

  4. Kaern M, Elston TC, Blake WJ, Collins JJ (2005) Stochasticity in gene expression: from theories to phenotypes. Nat Rev Genet 6:451–464

    Article  CAS  Google Scholar 

  5. Elowitz MB, Levine AJ, Siggia ED, Swain PS (2002) Stochastic gene expression in a single cell. Science 297:1183–1186

    Article  CAS  Google Scholar 

  6. Elowitz MB, Leibler S (2000) A synthetic oscillatory network of transcriptional regulators. Nature 403:335–338

    Article  CAS  Google Scholar 

  7. Silva-Rocha R, de Lorenzo V (2010) Noise and robustness in prokaryotic regulatory networks. Ann Rev Microbiol 64:257–275

    Article  CAS  Google Scholar 

  8. Hooshangi S, Thiberge S, Weiss R (2005) Ultrasensitivity and noise propagation in a synthetic transcriptional cascade. Proc Natl Acad Sci U S A 102:3581–3586

    Article  CAS  Google Scholar 

  9. Hasty J, Collins JJ (2002) Translating the noise. Nat Genet 31:13–14

    Article  CAS  Google Scholar 

  10. Raj A, van Oudenaarden A (2008) Nature, nurture, or chance: stochastic gene expression and its consequences. Cell 135:216–226

    Article  CAS  Google Scholar 

  11. Zaslaver A, Bren A, Ronen M, Itzkovitz S, Kikoin I, Shavit S, Liebermeister W, Surette MG, Alon U (2006) A comprehensive library of fluorescent transcriptional reporters for Escherichia coli. Nat Methods 3:623–628

    Article  CAS  Google Scholar 

  12. Brehm-Stecher BF, Johnson EA (2004) Single-cell microbiology: tools, technologies, and applications. Microbiol Mol Biol Rev 68:538–559

    Article  CAS  Google Scholar 

  13. Kelly JR, Rubin AJ, Davis JH, Ajo-Franklin CM, Cumbers J, Czar MJ, de Mora K, Glieberman AL, Monie DD, Endy D (2009) Measuring the activity of BioBrick promoters using an in vivo reference standard. J Biol Eng 3:4

    Article  Google Scholar 

  14. Heim R, Cubitt AB, Tsien RY (1995) Improved green fluorescence. Nature 373:663–664

    Article  CAS  Google Scholar 

  15. Kohlmeier S, Mancuso M, Tecon R, Harms H, van der Meer JR, Wells M (2007) Bioreporters: gfp versus lux revisited and single-cell response. Biosens Bioelectron 22:1578–1585

    Article  CAS  Google Scholar 

  16. de Las Heras A, Carreno CA, Martinez-Garcia E, de Lorenzo V (2010) Engineering input/output nodes in prokaryotic regulatory circuits. FEMS Microbiol Rev 34:842–865

    Article  Google Scholar 

  17. Keasling JD (1999) Gene-expression tools for the metabolic engineering of bacteria. Trends Biotechnol 17:452–460

    Article  CAS  Google Scholar 

  18. Meighen EA (1991) Molecular biology of bacterial bioluminescence. Microbiol Rev 55:123–142

    Article  CAS  Google Scholar 

  19. Meighen EA (1993) Bacterial bioluminescence: organization, regulation, and application of the lux genes. FASEB J 7:1016–1022

    Article  CAS  Google Scholar 

  20. Benedetti IM et al (2012) Quantitative, non disruptive monitoring of transcription in single cells with a broad-host range GFP-luxCDABE dual reporter system. PLoS One 7:e52000

    Article  CAS  Google Scholar 

  21. Silva-Rocha R, Martinez-Garcia E, Calles B, Chavarria M, Arce-Rodriguez A, de Las Heras A, Paez-Espino AD, Durante-Rodriguez G, Kim J, Nikel PI, Platero R, de Lorenzo V (2012) The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic Acids Res 41:D666–D675

    Article  Google Scholar 

  22. Cowles CE, Nichols NN, Harwood CS (2000) BenR, a XylS homologue, regulates three different pathways of aromatic acid degradation in Pseudomonas putida. J Bacteriol 182:6339–6346

    Article  CAS  Google Scholar 

  23. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  24. de Lorenzo V, Timmis KN (1994) Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10-derived minitransposons. Methods Enzymol 235:386–405

    Article  CAS  Google Scholar 

  25. Ferri SR, Soly RR, Szittner RB, Meighen EA (1991) Structure and properties of luciferase from Photobacterium phosphoreum. Biochem Biophys Res Commun 176:541–548

    Article  CAS  Google Scholar 

  26. Canton B, Labno A, Endy D (2008) Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26:787–793

    Article  CAS  Google Scholar 

  27. Young JW, Locke JC, Altinok A, Rosenfeld N, Bacarian T, Swain PS, Mjolsness E, Elowitz MB (2012) Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy. Nat Protoc 7:80–88

    Article  CAS  Google Scholar 

  28. Czechowska K, Johnson DR, van der Meer JR (2008) Use of flow cytometric methods for single-cell analysis in environmental microbiology. Curr Opin Microbiol 11:205–212

    Article  CAS  Google Scholar 

  29. Miller WG, Lindow SE (1997) An improved GFP cloning cassette designed for prokaryotic transcriptional fusions. Gene 191:149–153

    Article  CAS  Google Scholar 

  30. Herrero M, de Lorenzo V, Timmis KN (1990) Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol 172:6557–6567

    Article  CAS  Google Scholar 

  31. Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459–472

    Article  CAS  Google Scholar 

  32. Bagdasarian M, Lurz R, Ruckert B, Franklin FC, Bagdasarian MM, Frey J, Timmis KN (1981) Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene 16:237–247

    Article  CAS  Google Scholar 

  33. Keen NT, Tamaki S, Kobayashi D, Trollinger D (1988) Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene 70:191–197

    Article  CAS  Google Scholar 

  34. Antoine R, Locht C (1992) Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from gram-positive organisms. Mol Microbiol 6:1785–1799

    Article  CAS  Google Scholar 

  35. Thomas CM, Cross MA, Hussain AA, Smith CA (1984) Analysis of copy number control elements in the region of the vegetative replication origin of the broad host range plasmid RK2. EMBO J 3:57–63

    Article  CAS  Google Scholar 

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Acknowledgments

Authors are indebted to María Carmen Ortiz Navarro for help in flow cytometry assays. This work was supported by the BIO and FEDER CONSOLIDER-INGENIO programs of the Spanish Ministry of Economy and Competitiveness, the ARISYS and ST-FLOW Contracts of the EU, the ERANET-IB Program and funding from the Autonomous Community of Madrid (PROMPT). IB is the holder of an International Ph.D. Fellowship of La Caixa. All plasmids and strains described here are available upon request.

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Authors and Affiliations

  1. Systems Biology Program, Centro Nacional de Biotecnologia, CSIC, C/Darwin, 3 (Campus de Cantoblanco), Madrid, 28049, Spain

    Ilaria Benedetti & Victor de Lorenzo

Authors
  1. Ilaria Benedetti
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  2. Victor de Lorenzo
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Correspondence to Victor de Lorenzo .

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Editors and Affiliations

  1. MRC Centre for Molecular Bacteriology and Infection (CMBI) Department of Life Sciences, Imperial College, London, UK

    Alain Filloux

  2. Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain

    Juan-Luis Ramos

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Benedetti, I., de Lorenzo, V. (2014). Promoter Fusions with Optical Outputs in Individual Cells and in Populations. In: Filloux, A., Ramos, JL. (eds) Pseudomonas Methods and Protocols. Methods in Molecular Biology, vol 1149. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-0473-0_44

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  • DOI: https://doi.org/10.1007/978-1-4939-0473-0_44

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-0472-3

  • Online ISBN: 978-1-4939-0473-0

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