Stable implantation of orthogonal sensor circuits in Gram‐negative bacteria for environmental release

Abstract

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.

Increasing Signal Specificity of the TOL Network of Pseudomonas putida mt-2 by Rewiring the Connectivity of the Master Regulator XylR

“…To construct the corresponding mini-Tn 7 delivery vectors, we first engineered a series of pUC18Not derivatives [54] carrying the DNA segments at stake as follows. A 2.8-kb Kpn I- Sac I fragment of pBBXylR [56] containing the construct Pr → xylR (i.e., the xylR gene expressed through its native promoter of the TOL plasmid) was inserted in a pUC18Not variant that lacked the Eco RI site, thereby generating pBXe. This plasmid was used as the frame for replacing the native promoter region of xylR with the 3 alternative 5′-upstream sequences employed in this work.…”

“…Insertion of the corresponding segments in the naturally occurring att Tn 7 site of P. putida Pu·LUX was confirmed through colony PCR using a primer that anneals within the sequence of the B domain of xylR (NDoCAvrII: 5′-GCGAATGGCCTAGG CCGTAATACTG-3′) and one at the att Tn 7 insertion site within the glmS gene (Ppu glmS 2R: 5′-GTGCGTGCCCGTGGTGG-3′). The ensuing collection of Gm R strains were deleted of this antibiotic marker by transient expression of yeast flippase encoded by the plasmid pBBFLP, which brings about site-specific recombination of the FRT sequences that flank the resistance gene [56]. The same strategy was followed in the case of pTn7-BX17 and pTn7-Ps·RBX17, although the Gm R marker was not removed in these cases.…”

Environmental biosafety in the age of Synthetic Biology: Do we really need a radical new approach?

“…This may require a degree of adaptation of the synthetic genes to the new host that needs optimization through random mutations. The alternative is that the proteins/functions entered in the re-factored microorganism are designed in a fashion that makes their performance independent of the host [37,38]. It is more likely that larger and larger portions of natural genomic sequences will become replaced by synthetic DNA with engineered functions rather than having to re-synthesize the whole genome every time.…”

Modified 3-Oxoadipate Pathway for the Biodegradation of Methylaromatics inPseudomonas reinekeiMT1

“…The transconjugants generated by single crossover were selected on ABC medium supplemented with Gm, and merodiploids were resolved by additional plating on ABC medium supplemented with 5% sucrose. Deletion of the Gm r -GFP cassette was achieved by conjugation of the Flp-expressing pBBFLP plasmid (19) into the resulting strains by biparental mating using E. coli CC118pir (30) Complementation of MT1⌬mmlL mutant. The MT1⌬mmlL deletion mutant was separately complemented with the mmlL genes from P. reinekei MT1 and C. necator JMP134.…”