title: A set of versatile brick vectors and promoters for the assembly, expression, and integration of synthetic operons in Methylobacterium extorquens AM1 and other alphaproteobacteria.
abstract: The discipline of synthetic biology requires standardized tools and genetic elements to construct novel functionalities in microorganisms; yet, many model systems still lack such tools. Here, we describe a novel set of vectors that allows the convenient construction of synthetic operons in Methylobacterium extorquens AM1, an important alphaproteobacterial model organism for methylotrophy and a promising platform organism for methanol-based biotechnology. In addition, we provide a set of constitutive alphaproteobacterial promoters of different strengths that were characterized in detail by two approaches: on the single-cell scale and on the cell population level. Finally, we describe a straightforward strategy to deliver synthetic constructs to the genome of M. extorquens AM1 and other Alphaproteobacteria. This study defines a new standard to systematically characterize genetic parts for their use in M. extorquens AM1 by using single-cell fluorescence microscopy and opens the toolbox for synthetic biological applications in M. extorquens AM1 and other alphaproteobacterial model systems.
authors.affiliation: Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
authors.forename: Lennart
authors.initials: L
authors.lastname: Schada von Borzyskowski
authors.affiliation: Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
authors.forename: Mitja
authors.initials: M
authors.lastname: Remus-Emsermann
authors.affiliation: Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
authors.forename: Ramon
authors.initials: R
authors.lastname: Weishaupt
authors.affiliation: Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
authors.forename: Julia A
authors.initials: JA
authors.lastname: Vorholt
authors.affiliation: Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
authors.forename: Tobias J
authors.initials: TJ
authors.lastname: Erb
mesh_terms: D005822:Genetic Vectors / Q000235:genetics*; D020580:Methylobacterium / Q000235:genetics* / Q000378:metabolism; D009876:Operon; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014169:Transformation, Bacterial
chemical_list: D011994:Recombinant Proteins
keywords: Agrobacterium; Alphaproteobacteria; Caulobacter; Paracoccus; biobricks; operon assembly; promoter library; vector system