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A GFP-lacZ Bicistronic Reporter System for Promoter Analysis in Environmental Gram-Negative Bacteria
Here, we describe a bicistronic reporter system for the analysis of promoter activity in a variety of Gram-negative bacteria at both the population and single-cell levels. This synthetic genetic tool utilizes an artificial operon comprising the gfp and lacZ genes that are assembled in a suicide vect...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3321037/ https://www.ncbi.nlm.nih.gov/pubmed/22493710 http://dx.doi.org/10.1371/journal.pone.0034675 |
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author | Silva-Rocha, Rafael de Lorenzo, Victor |
author_facet | Silva-Rocha, Rafael de Lorenzo, Victor |
author_sort | Silva-Rocha, Rafael |
collection | PubMed |
description | Here, we describe a bicistronic reporter system for the analysis of promoter activity in a variety of Gram-negative bacteria at both the population and single-cell levels. This synthetic genetic tool utilizes an artificial operon comprising the gfp and lacZ genes that are assembled in a suicide vector, which is integrated at specific sites within the chromosome of the target bacterium, thereby creating a monocopy reporter system. This tool was instrumental for the complete in vivo characterization of two promoters, Pb and Pc, that drive the expression of the benzoate and catechol degradation pathways, respectively, of the soil bacterium Pseudomonas putida KT2440. The parameterization of these promoters in a population (using β-galactosidase assays) and in single cells (using flow cytometry) was necessary to examine the basic numerical features of these systems, such as the basal and maximal levels and the induction kinetics in response to an inducer (benzoate). Remarkably, GFP afforded a view of the process at a much higher resolution compared with standard lacZ tests; changes in fluorescence faithfully reflected variations in the transcriptional regimes of individual bacteria. The broad host range of the vector/reporter platform is an asset for the characterization of promoters in different bacteria, thereby expanding the diversity of genomic chasses amenable to Synthetic Biology methods. |
format | Online Article Text |
id | pubmed-3321037 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-33210372012-04-10 A GFP-lacZ Bicistronic Reporter System for Promoter Analysis in Environmental Gram-Negative Bacteria Silva-Rocha, Rafael de Lorenzo, Victor PLoS One Research Article Here, we describe a bicistronic reporter system for the analysis of promoter activity in a variety of Gram-negative bacteria at both the population and single-cell levels. This synthetic genetic tool utilizes an artificial operon comprising the gfp and lacZ genes that are assembled in a suicide vector, which is integrated at specific sites within the chromosome of the target bacterium, thereby creating a monocopy reporter system. This tool was instrumental for the complete in vivo characterization of two promoters, Pb and Pc, that drive the expression of the benzoate and catechol degradation pathways, respectively, of the soil bacterium Pseudomonas putida KT2440. The parameterization of these promoters in a population (using β-galactosidase assays) and in single cells (using flow cytometry) was necessary to examine the basic numerical features of these systems, such as the basal and maximal levels and the induction kinetics in response to an inducer (benzoate). Remarkably, GFP afforded a view of the process at a much higher resolution compared with standard lacZ tests; changes in fluorescence faithfully reflected variations in the transcriptional regimes of individual bacteria. The broad host range of the vector/reporter platform is an asset for the characterization of promoters in different bacteria, thereby expanding the diversity of genomic chasses amenable to Synthetic Biology methods. Public Library of Science 2012-04-06 /pmc/articles/PMC3321037/ /pubmed/22493710 http://dx.doi.org/10.1371/journal.pone.0034675 Text en Silva-Rocha and de Lorenzo. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Silva-Rocha, Rafael de Lorenzo, Victor A GFP-lacZ Bicistronic Reporter System for Promoter Analysis in Environmental Gram-Negative Bacteria |
title | A GFP-lacZ Bicistronic Reporter System for Promoter Analysis in Environmental Gram-Negative Bacteria |
title_full | A GFP-lacZ Bicistronic Reporter System for Promoter Analysis in Environmental Gram-Negative Bacteria |
title_fullStr | A GFP-lacZ Bicistronic Reporter System for Promoter Analysis in Environmental Gram-Negative Bacteria |
title_full_unstemmed | A GFP-lacZ Bicistronic Reporter System for Promoter Analysis in Environmental Gram-Negative Bacteria |
title_short | A GFP-lacZ Bicistronic Reporter System for Promoter Analysis in Environmental Gram-Negative Bacteria |
title_sort | gfp-lacz bicistronic reporter system for promoter analysis in environmental gram-negative bacteria |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3321037/ https://www.ncbi.nlm.nih.gov/pubmed/22493710 http://dx.doi.org/10.1371/journal.pone.0034675 |
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