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A comprehensive library of fluorescent transcriptional reporters for Escherichia coli


E. coli is widely used for systems biology research; there exists a need, however, for tools that can be used to accurately and comprehensively measure expression dynamics in individual living cells. To address this we present a library of transcriptional fusions of gfp to each of about 2,000 different promoters in E. coli K12, covering the great majority of the promoters in the organism. Each promoter fusion is expressed from a low-copy plasmid. We demonstrate that this library can be used to obtain highly accurate dynamic measurements of promoter activity on a genomic scale, in a glucose-lactose diauxic shift experiment. The library allowed detection of about 80 previously uncharacterized transcription units in E. coli, including putative internal promoters within previously known operons, such as the lac operon. This library can serve as a tool for accurate, high-resolution analysis of transcription networks in living E. coli cells.

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Figure 1: High-throughput cloning strategy used for the construction of the reporter-strain library, and promoter activity of the reporter strains during a diauxic shift experiment.
Figure 2: Gene expression dynamic profiles of reporter strains from diverse systems as measured during the diauxic shift experiment.
Figure 3: Identification of previously uncharacterized promoters.


  1. Alon, U. An introduction to systems biology: design principles of biological circuits (CRC press, USA, 2006).

    Google Scholar 

  2. Brown, P.O. & Botstein, D. Exploring the new world of the genome with DNA microarrays. Nat. Genet. 21, 33–37 (1999).

    Article  CAS  Google Scholar 

  3. Dharmadi, Y. & Gonzalez, R. DNA microarrays: experimental issues, data analysis, and application to bacterial systems. Biotechnol. Prog. 20, 1309–1324 (2004).

    Article  CAS  Google Scholar 

  4. Southward, C. & Surette, M.G. The dynamic microbe: green fluorescent protein brings bacteria to light. Mol. Microbiol. 45, 1191–1196 (2002).

    Article  CAS  Google Scholar 

  5. Cormack, B.P., Valdivia, R.H. & Falkow, S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38 (1996).

    Article  CAS  Google Scholar 

  6. Ronen, M., Rosenberg, R., Shraiman, B.I. & Alon, U. Assigning numbers to the arrows: parameterizing a gene regulation network by using accurate expression kinetics. Proc. Natl. Acad. Sci. USA 99, 10555–10560 (2002).

    Article  CAS  Google Scholar 

  7. Friedman, N., Vardi, S., Ronen, M., Alon, U. & Stavans, J. Precise temporal modulation in the response of the SOS DNA repair network in individual bacteria. PLoS Biol. 3, e238 (2005).

    Article  Google Scholar 

  8. Zaslaver, A. et al. Just-in-time transcription program in metabolic pathways. Nat. Genet. 36, 486–491 (2004).

    Article  CAS  Google Scholar 

  9. Kalir, S. & Alon, U. Using a quantitative blueprint to reprogram the dynamics of the flagella gene network. Cell 117, 713–720 (2004).

    Article  CAS  Google Scholar 

  10. Kalir, S., Mangan, S. & Alon, U. A coherent feed-forward loop with a sum input function prolongs flagella expression in Escherichia coli. Mol. Syst. Biol. 1, 2005. 0006 (2005).

  11. Kalir, S. et al. Ordering genes in a flagella pathway by analysis of expression kinetics from living bacteria. Science 292, 2080–2083 (2001).

    Article  CAS  Google Scholar 

  12. Larrainzar, E., O'Gara, F. & Morrissey, J.P. Applications of autofluorescent proteins for in situ studies in microbial ecology. Annu. Rev. Microbiol. 59, 257–277 (2005).

    Article  CAS  Google Scholar 

  13. Shen-Orr, S.S., Milo, R., Mangan, S. & Alon, U. Network motifs in the transcriptional regulation network of Escherichia coli. Nat. Genet. 31, 64–68 (2002).

    Article  CAS  Google Scholar 

  14. Mangan, S., Zaslaver, A. & Alon, U. The coherent feedforward loop serves as a sign-sensitive delay element in transcription networks. J. Mol. Biol. 334, 197–204 (2003).

    Article  CAS  Google Scholar 

  15. Mangan, S., Itzkovitz, S., Zaslaver, A. & Alon, U. The incoherent feed-forward loop accelerates the response-time of the gal system of Escherichia coli. J. Mol. Biol. 356, 1073–1081 (2006).

    Article  CAS  Google Scholar 

  16. Setty, Y., Mayo, A.E., Surette, M.G. & Alon, U. Detailed map of a cis-regulatory input function. Proc. Natl. Acad. Sci. USA 100, 7702–7707 (2003).

    Article  CAS  Google Scholar 

  17. Mayo, A.E., Setty, Y., Chalamish, S., Zaslaver, A. & Alon, U. Plasticity of the cis-regulatory input function of a gene. PLoS Biol. 4, e45 (2006).

    Article  Google Scholar 

  18. Blattner, F.R. et al. The complete genome sequence of Escherichia coli K-12. Science 277, 1453–1474 (1997).

    Article  CAS  Google Scholar 

  19. Chang, D.E., Smalley, D.J. & Conway, T. Gene expression profiling of Escherichia coli growth transitions: an expanded stringent response model. Mol. Microbiol. 45, 289–306 (2002).

    Article  CAS  Google Scholar 

  20. Monod, J., Cohen-Bazire, G. & Cohn, M. The biosynthesis of beta-galactosidase (lactase) in Escherichia coli; the specificity of induction. Biochim. Biophys. Acta 7, 585–599 (1951).

    Article  CAS  Google Scholar 

  21. Hengge-Aronis, R. Integration of control devices: a global regulatory network in Escherichia coli. In Prokaryotic Gene Expression (ed., Baumberg, S.), 169–193 (Oxford University Press, New York, 1999).

    Google Scholar 

  22. Keseler, I.M. et al. EcoCyc: a comprehensive database resource for Escherichia coli. Nucleic Acids Res. 33, D334–D337 (2005).

    Article  CAS  Google Scholar 

  23. Liu, X. & Matsumura, P. Differential regulation of multiple overlapping promoters in flagellar class II operons in Escherichia coli. Mol. Microbiol. 21, 613–620 (1996).

    Article  CAS  Google Scholar 

  24. Mytelka, D.S. & Chamberlin, M.J. Escherichia coli fliAZY operon. J. Bacteriol. 178, 24–34 (1996).

    Article  CAS  Google Scholar 

  25. Ikebe, T., Iyoda, S. & Kutsukake, K. Structure and expression of the fliA operon of Salmonella typhimurium. Microbiol. 145, 1389–1396 (1999).

    Article  CAS  Google Scholar 

  26. Salgado, H. et al. The comprehensive updated regulatory network of Escherichia coli K-12. BMC Bioinformatics 7, 5 (2006).

    Article  Google Scholar 

  27. Ghaemmaghami, S. et al. Global analysis of protein expression in yeast. Nature 425, 737–741 (2003).

    Article  CAS  Google Scholar 

  28. Newman, J.R. et al. Single-cell proteomic analysis of S. cerevisiae reveals the architecture of biological noise. Nature 441, 840–846 (2006).

    Article  CAS  Google Scholar 

  29. Kaern, M., Elston, T.C., Blake, W.J. & Collins, J.J. Stochasticity in gene expression: from theories to phenotypes. Nat. Rev. Genet. 6, 451–464 (2005).

    Article  CAS  Google Scholar 

  30. Elowitz, M.B., Levine, A.J., Siggia, E.D. & Swain, P.S. Stochastic gene expression in a single cell. Science 297, 1183–1186 (2002).

    Article  CAS  Google Scholar 

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We thank R. Rosenberg, H. Sberro, I. Alaluf, P. Bashkin, K. Pabbaraju and J. McClure for assistance, M.B. Elowitz, S. Falkow, S. Leibler, B. Wanner and all members of our laboratories for discussions. We thank the US National Institutes of Health, the Israel Science Foundation, Minerva, the Human Frontier Science Program and the Kahn Family Foundation for support.

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Correspondence to Uri Alon.

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Supplementary information

Supplementary Fig. 1

Distribution of intergenic regions length in E. coli, and the distribution of the length of the promoter regions used to construct the library. (PDF 44 kb)

Supplementary Fig. 2

Representative 100 strains measured during a diauxic shift experiment. (PDF 156 kb)

Supplementary Fig. 3

mRNA levels of fliY and fliA as measured by realtime PCR. (PDF 77 kb)

Supplementary Fig. 4

Expression profiles of wild type and mutant fliY reporter strains. (PDF 74 kb)

Supplementary Fig. 5

Real-time PCR and expression profiles of lacY and lacZ. (PDF 70 kb)

Supplementary Note (PDF 137 kb)

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Zaslaver, A., Bren, A., Ronen, M. et al. A comprehensive library of fluorescent transcriptional reporters for Escherichia coli. Nat Methods 3, 623–628 (2006).

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