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.