INSERTION MUTAGENESIS WITH Tn917-LTV1 IN THE GENOMIC DNA OF Bacillus anthracis

Abstract

The original intention of this study was to use the transposition element Tn917 as a tool to map promoters in the L1mes-1 strain of Bacillus anthracis (B. anthracis). The tranposable element Tn917 was introduced into the genome of B. anthracis strain 6Ames via plasmid pLTV1. Several related studies were designed and performed to analyze the proficiency of transposition, randomness of insertion, and any physiological effects of integration at particular sites. The temperaturesensitive plasmid was heat-cured from transformed &Ames clones and genomic DNA containing inserts at various sites, herein designated TnAmes, was extracted from survivors selectively cultured on erythromycin agar plates. Southern blot analysis confirmed that each DNA preparation contained Tn917 insertions. TnAmes DNA was then digested with EcoR1 restriction endonuclease, ligated, and used to transform Escherichia coli (E. coli) strain DH1OB electrocompetent cells.