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Nucleic Acids Res. 2015 Jul 27;43(13):e83. doi: 10.1093/nar/gkv248. Epub 2015 Mar 23.

A set of powerful negative selection systems for unmodified Enterobacteriaceae.

Author information

1
National University of Singapore, Department of Medicine, Yong Loo Lin School of Medicine, 1E Kent Ridge Road, NUHS Tower Block, Level 10, Singapore 119074.
2
National University of Singapore, Department of Medicine, Yong Loo Lin School of Medicine, 1E Kent Ridge Road, NUHS Tower Block, Level 10, Singapore 119074 Genome Institute of Singapore, Infectious Diseases Group, 60 Biopolis Street, Genome, #02-01, Singapore 138672 slchen@gis.a-star.edu.sg.

Abstract

Creation of defined genetic mutations is a powerful method for dissecting mechanisms of bacterial disease; however, many genetic tools are only developed for laboratory strains. We have designed a modular and general negative selection strategy based on inducible toxins that provides high selection stringency in clinical Escherichia coli and Salmonella isolates. No strain- or species-specific optimization is needed, yet this system achieves better selection stringency than all previously reported negative selection systems usable in unmodified E. coli strains. The high stringency enables use of negative instead of positive selection in phage-mediated generalized transduction and also allows transfer of alleles between arbitrary strains of E. coli without requiring phage. The modular design should also allow further extension to other bacteria. This negative selection system thus overcomes disadvantages of existing systems, enabling definitive genetic experiments in both lab and clinical isolates of E. coli and other Enterobacteriaceae.

PMID:
25800749
PMCID:
PMC4513841
DOI:
10.1093/nar/gkv248
[Indexed for MEDLINE]
Free PMC Article

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