Stable implantation of orthogonal sensor circuits in Gram-negative bacteria for environmental release.
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 Tn5-mini-transposon bearing an optimal attTn7 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.
de las Heras
Aitor
A
Centro Nacional de Biotecnología, CSIC. Darwin 3, Cantoblanco, 28049 Madrid, Spain.
Carreño
Carlos A
CA
de Lorenzo
Víctor
V
D015374:Biosensing Techniques / Q000379:methods*; D004251:DNA Transposable Elements; D004136:Dinitrobenzenes / Q000378:metabolism; D053834:Explosive Agents / Q000378:metabolism; D008027:Light; D010957:Plasmids; D016958:Pseudomonas putida / Q000235:genetics* / Q000378:metabolism*
D004251:DNA Transposable Elements; D004136:Dinitrobenzenes; D053834:Explosive Agents; C016403:2,4-dinitrotoluene