Development of new positive-selection RIVET tools: detection of induced promoters by the excision-based transcriptional activation of an aacCI (GmR)-gfp fusion.
J Biotechnol
; 155(2): 147-55, 2011 Sep 10.
Article
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| MEDLINE
| ID: mdl-21723338
ABSTRACT
RIVET (Recombination Based in vivo Expression Technology) is a powerful genetic tool originally conceived for the identification of genes induced in complex biological niches where conventional transcriptomics is difficult to use. With a broader application, genetic recombination-based technologies have also been used, in combination with regulatory proteins and specific transcriptional regulators, for the development of highly sensitive biosensor systems. RIVET systems generally comprise two modules a promoter-trap cassette generating genomic transcriptional fusions to the tnpR gene encoding the Tn-γδ TnpR resolvase, and a reporter cassette carrying res-flanked selection markers that are excised upon expression of tnpR to produce an irreversible, inheritable phenotypic change. We report here the construction and validation of a new set of positive-selection RIVET systems that, upon induction of the promoter-trap module, generate the transcriptional activation of an antibiotic-resistant and a green-fluorescent phenotype. Two classes of promoter-trap tools were constructed to generate transcriptional fusions to tnpR one based on the use of a narrow-host-range plasmid (pRIVET-I), integrative in several Gram-negative bacteria, and the other based on the use of a broad-host-range plasmid (pRIVET-R). The system was evaluated in the model soil bacterium Sinorhizobium meliloti, where a clear-cut phenotypic transition from Nm(R)-Gm(S)-GFP(-) to Nm(S)-Gm(R)-GFP(+) occurred upon expression of tnpR. A S. meliloti integrative RIVET library was constructed in pRIVET-I and, as expected, changes in the extracellular conditions (e.g., salt stress) triggered a significant increase in the appearance of Gm(R)-GFP(+) (excised) clones. The sacB-independent positive-selection RIVET systems here described provide suitable basic tools both for the construction of new recombination-based biosensors and for the search of bacterial markers induced when microorganisms colonize and invade complex environments and eukaryotic hosts.
Texto completo:
1
Base de datos:
MEDLINE
Asunto principal:
Recombinación Genética
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Proteínas Recombinantes de Fusión
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Técnicas Biosensibles
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Regulación Bacteriana de la Expresión Génica
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Activación Transcripcional
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Sinorhizobium meliloti
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Perfilación de la Expresión Génica
Tipo de estudio:
Diagnostic_studies
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Evaluation_studies
Idioma:
En
Revista:
J Biotechnol
Asunto de la revista:
BIOTECNOLOGIA
Año:
2011
Tipo del documento:
Article
País de afiliación:
Argentina