Site-Directed Genome Integration via Recombinase-Mediated Cassette Exchange (RMCE) in Escherichia coli

Abstract

The gold standard for successful genome integration in Escherichia coli is the homologous recombination by the bacteriophage-inspired lambda Red system. This method uses the bacteriophage lambda Red recombination proteins to promote homologous recombination between a target DNA sequence and a DNA fragment, which is introduced into the bacterial cell by electroporation. It allows researchers to create specific genetic changes in bacterial genomes, making it a valuable tool for studies in microbiology and biotechnology. However, this system is not without limitations, which are characteristic of its working mechanism and remain to present challenges. The most formidable constraints stem from nucleotide sequences that contain self-homology or homologies to the host genome. These instances lead to uncontrolled homologous recombination events, consequently hindering the desired integration event. Furthermore, handling very large fragments can also be problematic, although, in many instances, this can be overcome by multiple lambda Red integrations in a row. In this study, we illustrate that the limitations associated with the lambda Red system can be overcome through the application of recombinase-mediated cassette exchange (RMCE). This enables the genome integration of larger and more complex DNA fragments and facilitates new research opportunities.