Development of a genome swapping method using a Cre/lox recombination system

Abstract

The functional genomics and comparative genomics of last decade have revealed new genome information of various organisms and identified essential genes in genome and functions of genes. And a significant goal in post-genome era is the construction of an artificial microorganism. In order to accelerate the construction of an artificial microorganism, an efficient genomic engineering tool which can transfer large genomic segments carrying useful genomic information is needed. We developed a novel and efficient method called ‘genome swapping’ using Cre/lox site-specific recombination system and γ-ori replication system to transfer the large genomic segments among various microorganisms. The Genome swapping begins by in vivo excision and amplification of large genomic segments of donor genome by Cre/lox site-specific recombination system and γ-ori replication system. These amplified genomic segments are linearized by the meganuclease I-SceI and transferred into recipient cells. By Recombinase mediated cassette exchange using Cre/lox site-specific recombination system, the amplified genomic segment of donor genome is replaced for the recipient genome. Using the genome swapping method, the large genomic segments up to 1.1Mb, carrying deleted regions of donor genome, were successfully excised and amplified. This genome swapping method should be useful for the construction of the minimized genome or hybrid artificial microorganisms.