Targeted allele replacement mutagenesis of Corynebacterium pseudotuberculosis

Caray A. Walker; Willie Donachie; David George Emslie Smith; Michael C. Fontaine

doi:10.1128/AEM.01740-10

Targeted allele replacement mutagenesis of Corynebacterium pseudotuberculosis

Caray A. Walker, Willie Donachie, David George Emslie Smith, Michael C. Fontaine

Research output: Contribution to journal › Article › peer-review

2 Citations (Scopus)

Abstract

A two-step allele replacement mutagenesis procedure, using a conditionally replicating plasmid, was developed to allow the creation of targeted, marker-free mutations in Corynebacterium pseudotuberculosis. The relationship between homologous sequence length and recombination frequency was determined, and enhanced plasmid excision was observed due to the rolling-circle replication of the mutagenesis vector. Furthermore, an antibiotic enrichment procedure was applied to improve the recovery of mutants. Subsequently, as proof of concept, a marker-free, cp40-deficient mutant of C. pseudotuberculosis was constructed.

Original language	English
Pages (from-to)	3532-3535
Number of pages	4
Journal	Applied and Environmental Microbiology
Volume	77
Issue number	10
DOIs	https://doi.org/10.1128/AEM.01740-10
Publication status	Published - May 2011

Keywords

Alleles
Anti-Bacterial Agents
Corynebacterium pseudotuberculosis
DNA, Bacterial
Gene Deletion
Gene Targeting
Genetic Vectors
Molecular Sequence Data
Mutagenesis, Insertional
Plasmids
Selection, Genetic
Sequence Analysis, DNA

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10.1128/AEM.01740-10

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title = "Targeted allele replacement mutagenesis of Corynebacterium pseudotuberculosis",

abstract = "A two-step allele replacement mutagenesis procedure, using a conditionally replicating plasmid, was developed to allow the creation of targeted, marker-free mutations in Corynebacterium pseudotuberculosis. The relationship between homologous sequence length and recombination frequency was determined, and enhanced plasmid excision was observed due to the rolling-circle replication of the mutagenesis vector. Furthermore, an antibiotic enrichment procedure was applied to improve the recovery of mutants. Subsequently, as proof of concept, a marker-free, cp40-deficient mutant of C. pseudotuberculosis was constructed.",

keywords = "Alleles, Anti-Bacterial Agents, Corynebacterium pseudotuberculosis, DNA, Bacterial, Gene Deletion, Gene Targeting, Genetic Vectors, Molecular Sequence Data, Mutagenesis, Insertional, Plasmids, Selection, Genetic, Sequence Analysis, DNA",

author = "Walker, {Caray A.} and Willie Donachie and Smith, {David George Emslie} and Fontaine, {Michael C.}",

year = "2011",

month = may,

doi = "10.1128/AEM.01740-10",

language = "English",

volume = "77",

pages = "3532--3535",

journal = "Applied and Environmental Microbiology",

issn = "0099-2240",

publisher = "American Society for Microbiology",

number = "10",

}

TY - JOUR

T1 - Targeted allele replacement mutagenesis of Corynebacterium pseudotuberculosis

AU - Walker, Caray A.

AU - Donachie, Willie

AU - Smith, David George Emslie

AU - Fontaine, Michael C.

PY - 2011/5

Y1 - 2011/5

N2 - A two-step allele replacement mutagenesis procedure, using a conditionally replicating plasmid, was developed to allow the creation of targeted, marker-free mutations in Corynebacterium pseudotuberculosis. The relationship between homologous sequence length and recombination frequency was determined, and enhanced plasmid excision was observed due to the rolling-circle replication of the mutagenesis vector. Furthermore, an antibiotic enrichment procedure was applied to improve the recovery of mutants. Subsequently, as proof of concept, a marker-free, cp40-deficient mutant of C. pseudotuberculosis was constructed.

AB - A two-step allele replacement mutagenesis procedure, using a conditionally replicating plasmid, was developed to allow the creation of targeted, marker-free mutations in Corynebacterium pseudotuberculosis. The relationship between homologous sequence length and recombination frequency was determined, and enhanced plasmid excision was observed due to the rolling-circle replication of the mutagenesis vector. Furthermore, an antibiotic enrichment procedure was applied to improve the recovery of mutants. Subsequently, as proof of concept, a marker-free, cp40-deficient mutant of C. pseudotuberculosis was constructed.

KW - Alleles

KW - Anti-Bacterial Agents

KW - Corynebacterium pseudotuberculosis

KW - DNA, Bacterial

KW - Gene Deletion

KW - Gene Targeting

KW - Genetic Vectors

KW - Molecular Sequence Data

KW - Mutagenesis, Insertional

KW - Plasmids

KW - Selection, Genetic

KW - Sequence Analysis, DNA

U2 - 10.1128/AEM.01740-10

DO - 10.1128/AEM.01740-10

M3 - Article

C2 - 21421779

SN - 0099-2240

VL - 77

SP - 3532

EP - 3535

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

IS - 10

ER -

Targeted allele replacement mutagenesis of Corynebacterium pseudotuberculosis

Abstract

Keywords

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