Design-Build-Test of Synthetic Promoters for Inducible Gene Regulation in Alphaproteobacteria

Inducible gene expression is useful for biotechnological applications and for studying gene regulation and function in bacteria. Many inducible systems that perform in model organisms such as the Gammaproteobacterium Escherichia coli do not perform well in other bacteria that are of biotechnological interest. Typical problems include weak or leaky expression. Here, we describe an invention named ACIT (Alphaproteobacteria chromosomally integrating transcription-control cassette) that is carried on a suicide plasmid to enable insertion into the chromosome of the host. ACIT consists of multiple DNA fragments specifically arranged in a cassette that allows tight transcription control over any gene or gene cluster of interest following homologous recombination. At the heart of the invention is the ability to modify or exchange parts, e.g., promoters, to suit particular bacteria and growth conditions, allowing for customized gene expression control. Furthermore, ACIT provides a basis for a design-build-test approach for controlling gene expression in less studied bacteria. We describe examples of its control over pigment and exopolysaccharide production, growth, cell form, and social behavior in various Alphaproteobacteria.