Site-specific recombinases for integration of heterologous DNA in E. coli

Escherichia coli is an important model organism for bioengineering, but even it can be limited by the genetic tools available.  One method used to insert heterologous DNA into the E. coli chromosome is the conditional-replication, integration, and modular plasmid (CRIM) system, which uses site-specific DNA recombinases from native E. coli phages to integrate plasmids into one of five spots in the chromosome.  The ability to integrate more than five constructs is needed though, so we have expanded the CRIM system using site-specific serine integrases from other bacteriophage.  This group of integrases don’t require host proteins for functionality and thus function in phylogenetically diverse organisms. They enable a single, unidirectional recombination event between two specific DNA sequences, attB and attP, resulting in two new sites, attR and attL. Using these integrases, we have developed a system to stably integrate up to fourteen genetic constructs into in the E. coli genome.  Fourteen attB sites were added to chromosome at the HK phage site and the corresponding integrases were cloned onto temperature sensitive plasmids to mediate recombination between a plasmid containing an attP site and the corresponding attB site on the chromosome.  The attP-containing plasmid further contains the PhiC31 attachment sites flanking an antibiotic gene.  This fragment can then be removed by introducing the PhiC31 integrase, preventing the accumulation of duplicated DNA and allowing reuse of the antibiotic resistance gene.  This system improves upon existing genetic tools in E. coli and provides a road map for multiple gene integration in other organisms.