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General comments

A plasmid for the generating of gene inactivation mutants in naturally nontransformable gram-positive bacteria. This vector allows, on X-Gal (5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside) plates, a quick colorimetric blue-white discrimination of bacteria which have lost the plasmid, facilitating clone identification during mutagenesis. The plasmid is can be used in Staphylococcus aureus, Listeria monocytogenes, and Bacillus spp. to efficiently construct mutants with or without an associated antibiotic resistance gene.

  • E.coli: multicopy plasmid, select for ampicillin
  • B. subtilis: temperature sensitive ori, select for erythromycin/ lincomycin

Hints for the usage

  • for a markerless deletion of a gene amplify 1000 bp up- and downstream of the gene and clone them into the MCS of the plasmid

The reference

Maryvonne Arnaud, Arnaud Chastanet, Michel Débarbouillé
New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria.
Appl. Environ. Microbiol.: 2004, 70(11);6887-91
[PubMed:15528558] [] [DOI] (P p)