title: Promoter fusions with optical outputs in individual cells and in populations.
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.
authors.affiliation: Systems Biology Program, Centro Nacional de Biotecnologia, CSIC, C/Darwin, 3 (Campus de Cantoblanco), Madrid, 28049, Spain.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Benedetti
authors.forename: Victor
authors.initials: V
authors.lastname: de Lorenzo
mesh_terms: D019677:Artificial Gene Fusion / Q000379:methods*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005434:Flow Cytometry; D005453:Fluorescence; D005822:Genetic Vectors / Q000378:metabolism; D008163:Luminescent Measurements; D055620:Optical Phenomena; D010957:Plasmids; D011401:Promoter Regions, Genetic; D016958:Pseudomonas putida / Q000166:cytology* / Q000235:genetics*; D059010:Single-Cell Analysis
chemical_list: D004269:DNA, Bacterial