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Stable implantation of orthogonal sensor circuits in Gram‐negative bacteria for environmental release
Author(s) -
De Las Heras Aitor,
Carreño Carlos A.,
De Lorenzo Víctor
Publication year - 2008
Publication title -
environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.954
H-Index - 188
eISSN - 1462-2920
pISSN - 1462-2912
DOI - 10.1111/j.1462-2920.2008.01722.x
Subject(s) - biology , transposable element , plasmid , computational biology , reporter gene , genetics , homologous recombination , pseudomonas putida , microbiology and biotechnology , gene , genome , gene expression
Summary A broad host range, orthogonal genetic platform has been developed to format sensor circuits in the chromosome of Gram‐negative microorganisms destined for environmental release as bioindicators of toxic or perilous compounds (e.g. explosives) in soil. The genetic scheme includes the generation of a genomic landing pad for the sensor module with a Tn 5 ‐mini‐transposon bearing an optimal att Tn 7 sequence and a choice of reporter systems with optical and enzymatic outputs. The array of functional elements thereby inserted in the chromosome match that of a cognate plasmid vector which delivers the transcription factors and the promoters to a frame that places the regulatory parts in front of the reporters. Site‐specific recombination sites allow the deletion of antibiotic resistances and enables reporter output prior to deliberate release. The system thus allows the production and maintenance of cells in a pre‐release state and its intentional conversion in deliverable strains that fulfil all safety, stability and performance criteria. The combination of such a genetic platform with a variant of the transcriptional regulator XylR of Pseudomonas putida that responds to 2,4‐dinitrotoluene has been the basis for the production of strains that emit light upon exposure to residues of explosives in a soil microcosm.