Insertional Mutagenesis Protocol for Constructing Single or Sequential Mutations

Abstract

Genetic mutation enables the study of the function of specific genes, particularly when a mutant is compared against its isogenic parent. In Proteus mirabilis bacteria, traditional allelic exchange mutation is labor-intensive and has a high failure rate in some strains. Likewise, there is no working protocol for lambda red recombinase-based mutation in P. mirabilis. Here we describe an alternative method of insertional mutagenesis based on retargeting of group II introns. The protocol includes steps to generate single or multiple mutations, with the possibility to delete intervening sequences of DNA.