A set of versatile brick vectors and promoters for the assembly, expression, and integration of synthetic operons in Methylobacterium extorquens AM1 and other alphaproteobacteria.

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.