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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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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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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