inPOSE: a flexible toolbox for chromosomal cloning and amplification of bacterial transgenes

Abstract

Cloning genes and operons encoding heterologous functions in bacterial hosts is almost exclusively carried out today using plasmid vectors. This has multiple drawbacks, including the need for constant selection and the variation in copy numbers. Chromosomal integration of transgenes has always offered a viable alternative, however, to date it has been of limited use due to its tedious nature and to being limited often to a single copy. We introduce here a strategy that uses bacterial insertion sequences, the simplest autonomous transposable elements to insert and amplify genetic cargo into a bacterial chromosome. Transgene insertion can take place either as transposition or homologous recombination, and copy-number amplification is achieved using controlled copy-paste transposition. We display successful use of IS1 and IS3 for this purpose in Escherichia coli cells, using various selection markers. We demonstrate the insertion of selectable genes, an unselectable gene, and a five-gene operon in up to two copies in a single step. We continue with the amplification of the inserted cassette to double-digit copy numbers within two rounds of transposase induction and selection. Finally, we analyze the stability of the cloned genetic constructs in the lack of selection and find it to be superior to all investigated plasmid-based systems. Due to the ubiquitous nature of transposable elements, we believe that with proper design, this strategy can be adapted to numerous further bacterial species.