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cobA, a red fluorescent transcriptional reporter for Escherichia coli, yeast, and mammalian cells

Nature Biotechnology volume 17, pages 1175–1178 (1999)Cite this article

Abstract

We demonstrate the use of Propionibacterium freudenreichii uroporphyrinogen III methyltransferase (cobA) as a reporter of gene expression in Escherichia coli, fission yeast, and mammalian cells. Overexpression of cobA in cells resulted in bright red fluorescence that was visualized with standard fluorescence microscopy and fluorescence-activated cell sorting analysis at the single-cell level. As with green fluorescent protein (GFP), no addition of exogenous substrate was required. When expressed in Chinese hamster ovary cells from a bicistronic transcript, cobA and GFP gave rise to fluorescence signals of similar intensity. The bright red fluorescence generated by the cobA reporter promises a better signal-to-noise ratio than blue and green fluorescent reporter systems, as autofluorescence and light scattering of cells, media, and materials are reduced in the red wavelengths.

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Figure 1: Inducible IPTG CobA expression in E. coli.
Figure 2: Thiamine-controlled CobA expression in fission yeast.
Figure 3: CobA expression in CHO-K1 cells transiently transfected with pVgRxR and pcDNA3, pcDNA3–cobA, or pIND–cobA.
Figure 4: Coexpression of CobA and GFP2 from a bicistronic mRNA.
Figure 5: FACScan of CobA- and EGFP-expressing CHO cells.

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Acknowledgements

We would like to thank Dr. Charles A. Roessner for the gift of pISA417; Adriana Missano, Nicole Holzwarth, Nina Barth, and Ashley Hayes for technical assistance; and Dr. Francis Mueller and E. Kunitz for help using the spectrophotometer.

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  1. Stefan Wildt

    Present address: Massachusetts Institute of Technology, Chemical Engineering Department, 25 Ames St. B. 56-469, Cambridge, MA, 02139

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  1. Pharma Division, Preclinical CNS Research and GeneTechnologies Molecular Neurobiology, F. Hoffmann-La Roche AG, Basel, Switzerland

    Stefan Wildt & Ulrich Deuschle

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Correspondence to Ulrich Deuschle.

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Wildt, S., Deuschle, U. cobA, a red fluorescent transcriptional reporter for Escherichia coli, yeast, and mammalian cells. Nat Biotechnol 17, 1175–1178 (1999). https://doi.org/10.1038/70713

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