INVESTIGADORES
DRAGHI Walter Omar
artículos
Título:
Development of new positive-selection RIVET tools: detection of induced promoters by the excision-based transcriptional activation of an aacCI (GmR)-gfp fusion
Autor/es:
LOZANO, M.J.; SALAS, M.E.; GIUSTI, M.A.; DRAGHI, W.O.; TORRES TEJERIZO, G.A.; MARTINI, C.; DEL PAPA, M.F.; PISTORIO, M.; LAGARES, A.
Revista:
JOURNAL OF BIOTECHNOLOGY
Editorial:
ELSEVIER SCIENCE BV
Referencias:
Año: 2011 vol. 155 p. 147 - 155
ISSN:
0168-1656
Resumen:
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.