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• W2117278047 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." @default.
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• W2117278047 date "2011-09-01" @default.
• W2117278047 modified "2023-10-13" @default.
• W2117278047 title "Development of new positive-selection RIVET tools: Detection of induced promoters by the excision-based transcriptional activation of an aacCI (GmR)–gfp fusion" @default.
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• W2117278047 doi "https://doi.org/10.1016/j.jbiotec.2011.06.014" @default.
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