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A comprehensive library of fluorescent transcriptional reporters for Escherichia coli

Abstract

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.

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Acknowledgements

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). https://doi.org/10.1038/nmeth895

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