Quantitative, non-disruptive monitoring of transcription in single cells with a broad-host range GFP-luxCDABE dual reporter system.

A dual promoter probe system based on a tandem bi-cistronic GFP-luxCDABE reporter cassette is described and implemented. This system is assembled in two synthetic, modular, broad-host range plasmids based on pBBR1 and RK2 origins of replication, allowing its utilization in an extensive number of gra...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Ilaria Maria Benedetti, Victor de Lorenzo, Rafael Silva-Rocha
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/590e30c5590246b4b2333e292d93f826
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:590e30c5590246b4b2333e292d93f826
record_format	dspace
spelling	oai:doaj.org-article:590e30c5590246b4b2333e292d93f8262021-11-18T08:03:21ZQuantitative, non-disruptive monitoring of transcription in single cells with a broad-host range GFP-luxCDABE dual reporter system.1932-620310.1371/journal.pone.0052000https://doaj.org/article/590e30c5590246b4b2333e292d93f8262012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23284849/?tool=EBIhttps://doaj.org/toc/1932-6203A dual promoter probe system based on a tandem bi-cistronic GFP-luxCDABE reporter cassette is described and implemented. This system is assembled in two synthetic, modular, broad-host range plasmids based on pBBR1 and RK2 origins of replication, allowing its utilization in an extensive number of gram-negative bacteria. We analyze the performance of this dual cassette in two hosts, Escherichia coli and Pseudomonas putida, by examining the induction properties of the lacI(q)-Ptrc expression system in the first host and the Pb promoter of the benzoate degradation pathway in the second host. By quantifying the bioluminescence signal produced through the expression of the lux genes, we explore the dynamic range of induction for the two systems (Ptrc-based and Pb-based) in response to the two inducers. In addition, by quantifying the fluorescence signals produced by GFP expression, we were able to monitor the single-cell expression profile and to explore stochasticity of the same two promoters by flow cytometry. The results provided here demonstrate the power of the dual GFP-luxCDABE cassette as a new, single-step tool to assess promoter properties at both the population and single-cell levels in gram-negative bacteria.Ilaria Maria BenedettiVictor de LorenzoRafael Silva-RochaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 12, p e52000 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Ilaria Maria Benedetti Victor de Lorenzo Rafael Silva-Rocha Quantitative, non-disruptive monitoring of transcription in single cells with a broad-host range GFP-luxCDABE dual reporter system.
description	A dual promoter probe system based on a tandem bi-cistronic GFP-luxCDABE reporter cassette is described and implemented. This system is assembled in two synthetic, modular, broad-host range plasmids based on pBBR1 and RK2 origins of replication, allowing its utilization in an extensive number of gram-negative bacteria. We analyze the performance of this dual cassette in two hosts, Escherichia coli and Pseudomonas putida, by examining the induction properties of the lacI(q)-Ptrc expression system in the first host and the Pb promoter of the benzoate degradation pathway in the second host. By quantifying the bioluminescence signal produced through the expression of the lux genes, we explore the dynamic range of induction for the two systems (Ptrc-based and Pb-based) in response to the two inducers. In addition, by quantifying the fluorescence signals produced by GFP expression, we were able to monitor the single-cell expression profile and to explore stochasticity of the same two promoters by flow cytometry. The results provided here demonstrate the power of the dual GFP-luxCDABE cassette as a new, single-step tool to assess promoter properties at both the population and single-cell levels in gram-negative bacteria.
format	article
author	Ilaria Maria Benedetti Victor de Lorenzo Rafael Silva-Rocha
author_facet	Ilaria Maria Benedetti Victor de Lorenzo Rafael Silva-Rocha
author_sort	Ilaria Maria Benedetti
title	Quantitative, non-disruptive monitoring of transcription in single cells with a broad-host range GFP-luxCDABE dual reporter system.
title_short	Quantitative, non-disruptive monitoring of transcription in single cells with a broad-host range GFP-luxCDABE dual reporter system.
title_full	Quantitative, non-disruptive monitoring of transcription in single cells with a broad-host range GFP-luxCDABE dual reporter system.
title_fullStr	Quantitative, non-disruptive monitoring of transcription in single cells with a broad-host range GFP-luxCDABE dual reporter system.
title_full_unstemmed	Quantitative, non-disruptive monitoring of transcription in single cells with a broad-host range GFP-luxCDABE dual reporter system.
title_sort	quantitative, non-disruptive monitoring of transcription in single cells with a broad-host range gfp-luxcdabe dual reporter system.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/590e30c5590246b4b2333e292d93f826
work_keys_str_mv	AT ilariamariabenedetti quantitativenondisruptivemonitoringoftranscriptioninsinglecellswithabroadhostrangegfpluxcdabedualreportersystem AT victordelorenzo quantitativenondisruptivemonitoringoftranscriptioninsinglecellswithabroadhostrangegfpluxcdabedualreportersystem AT rafaelsilvarocha quantitativenondisruptivemonitoringoftranscriptioninsinglecellswithabroadhostrangegfpluxcdabedualreportersystem
_version_	1718422603436130304

Quantitative, non-disruptive monitoring of transcription in single cells with a broad-host range GFP-luxCDABE dual reporter system.

Ejemplares similares