title: Efficient switching of mycobacteriophage L5-based integrating plasmids in Mycobacterium tuberculosis.
abstract: We previously used a mycobacteriophage L5-derived integrating vector to demonstrate that glnE and aroK are essential genes in Mycobacterium tuberculosis by showing that we were unable to excise the integrated vector when it carried the only functional copy of these genes. We tested three systems to replace the integrated copy with alternative alleles. The most efficient method was to transform the strain with a second copy of the integrating vector. Excision of the resident vector and integration of the incoming vector occurred at an extremely high efficiency. This technique will allow us to study the role and functionality of essential genes in this important human pathogen.
authors.affiliation: Centre for Infectious Disease, Institute of Cell and Molecular Science, Barts and the London, Queen Mary's School of Medicine and Dentistry, London E1 2AD, UK.
authors.forename: Carey A
authors.initials: CA
authors.lastname: Pashley
authors.forename: Tanya
authors.initials: T
authors.lastname: Parish
mesh_terms: D000483:Alleles; D005816:Genetic Complementation Test; D005974:Glutamate-Ammonia Ligase / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000379:methods*; D009160:Mycobacteriophages / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000821:virology*; D010957:Plasmids / Q000235:genetics; D016662:Virus Integration
chemical_list: D005974:Glutamate-Ammonia Ligase