Construction of a new gene insertion tool on the Proteus mirabilis chromosome: mini-ICE

Abstract

Chromosomal insertion mechanisms are effective for genetic engineering and ideal for DNA integration in situations where plasmid selection is not feasible, such as in vaccine strains or for environmental release. The creation of new mechanisms for gene insertion into the chromosome is important to expand the range of gene expression systems, reach new bacterial species, and also to use different mechanisms together. This project proposes the use of integrative conjugative elements (ICEs) to construct a novel site-specific gene delivery mechanism on the chromosome, the mini-ICE. The mini-ICE is composed of DNA fragments that provide: a ICE insertion site, a multiple cloning site, and Flip recombinase enzyme sites. The Flip recombinase enzyme sites flank chloramphenicol resistance genes, replication origin, transfer origin, and genes encoding the integrase enzyme. The mini-ICE will be tested on Proteus mirabilis and subsequently on other bacteria of interest.(AU)