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Development of new positive-selection RIVET tools: Detection of induced promoters by the excision-based transcriptional activation of an aacCI (GmR)–gfp fusion

Paper ID Volume ID Publish Year Pages File Format Full-Text
23713 43467 2011 9 PDF Available
Title
Development of new positive-selection RIVET tools: Detection of induced promoters by the excision-based transcriptional activation of an aacCI (GmR)–gfp fusion
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 NmR-GmS-GFP− to NmS-GmR-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 GmR-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.

Keywords
RIVET; IVET; Positive-selection; Bacteria–host interaction; Sinorhizobium meliloti; Biosensor
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Development of new positive-selection RIVET tools: Detection of induced promoters by the excision-based transcriptional activation of an aacCI (GmR)–gfp fusion
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 155, Issue 2, 10 September 2011, Pages 147–155
Authors
, , , , , , , , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us