title: Tn7-Based Device for Calibrated Heterologous Gene Expression in Pseudomonas putida.
abstract: The soil bacterium Pseudomonas putida is increasingly attracting considerable interest as a platform for advanced metabolic engineering through synthetic biology approaches. However, genomic context, gene copy number, and transcription/translation interplay often introduce considerable uncertainty to the design of reliable genetic constructs. In this work, we have established a standardized heterologous expression device in which the promoter strength is the only variable; the remaining parameters of the flow have stable default values. To this end, we tailored a mini-Tn7 delivery transposon vector that inserts the constructs in a single genomic locus of P. putida's chromosome. This was then merged with a promoter insertion site, an unvarying translational coupler, and a downstream location for placing the gene(s) of interest under fixed assembly rules. This arrangement was exploited to benchmark a collection of synthetic promoters with low transcriptional noise in this bacterial host. Growth experiments and flow cytometry with single-copy promoter-GFP constructs revealed a robust, constitutive behavior of these promoters, whose strengths and properties could be faithfully compared. This standardized expression device significantly extends the repertoire of tools available for reliable metabolic engineering and other genetic enhancements of P. putida.
authors.affiliation: Institute of Applied Microbiology, RWTH Aachen University , Worringerweg 1, 52074 Aachen, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Zobel
authors.affiliation: Systems Biology Program, Centro Nacional de Biotecnologia, CSIC , C/Darwin, 3 (Campus de Cantoblanco), Madrid 28049, Spain.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Benedetti
authors.affiliation: Institute of Applied Microbiology, RWTH Aachen University , Worringerweg 1, 52074 Aachen, Germany.
authors.forename: Lara
authors.initials: L
authors.lastname: Eisenbach
authors.affiliation: Systems Biology Program, Centro Nacional de Biotecnologia, CSIC , C/Darwin, 3 (Campus de Cantoblanco), Madrid 28049, Spain.
authors.forename: Victor
authors.initials: V
authors.lastname: de Lorenzo
authors.affiliation: Institute of Applied Microbiology, RWTH Aachen University , Worringerweg 1, 52074 Aachen, Germany.
authors.forename: Nick
authors.initials: N
authors.lastname: Wierckx
authors.affiliation: Institute of Applied Microbiology, RWTH Aachen University , Worringerweg 1, 52074 Aachen, Germany.
authors.forename: Lars M
authors.initials: LM
authors.lastname: Blank
mesh_terms: D002876:Chromosomes, Bacterial / Q000235:genetics / Q000378:metabolism; D004251:DNA Transposable Elements / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics*; D016958:Pseudomonas putida; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D004251:DNA Transposable Elements; D011994:Recombinant Proteins
keywords: Pseudomonas putida; Tn7 transposon; bicistronic design; synthetic biology; synthetic promoters; translational coupler