Multicopy insAB Sequences Are Efficient Gene Integration Sites in the Escherichia coli Genome

Multiple replication sequences in a genome can provide multiple integration sites, thus increasing target gene integration efficiency. In this study, in the Escherichia coli BL21(DE3) genome, we identified 19 multicopy sequences including rDNA sites and insAB repeats that occurred 28 times and had the highest copy number. For target gene integration, at least 15/28 insAB sites underwent target gene integration with no concomitant effects on cell growth. In addition, lacZ activity at insAB sites reached approximately 4000 U/mg and was ten times that of rDNA sites. From our data, multicopy insAB sequences had favorable sites for target gene integration in E. coli. Therefore, using insAB sites, targeted gene expression in the E. coli genome can be conveniently adjusted to high levels.