Clostridium difficile Genome Editing Using pyrE Alleles

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Clostridium difficile Genome Editing Using pyrE Alleles. / Ehsaan, Muhammad; Kuehne, Sarah; Minton, Nigel P.

In: Methods in molecular biology, Vol. 1476, 10.08.2016, p. 35-52.

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Ehsaan, Muhammad ; Kuehne, Sarah ; Minton, Nigel P. / Clostridium difficile Genome Editing Using pyrE Alleles. In: Methods in molecular biology. 2016 ; Vol. 1476. pp. 35-52.

Bibtex

@article{5b97bf254f264ccc832dbef6bb7f4a25,
title = "Clostridium difficile Genome Editing Using pyrE Alleles",
abstract = "Precise manipulation (in-frame deletions and substitutions) of the Clostridium difficile genome is possible through a two-stage process of single-crossover integration and subsequent isolation of double-crossover excision events using replication-defective plasmids that carry a counterselection marker. Use of a codA (cytosine deaminase) or pyrE (orotate phosphoribosyltransferase) as counter selection markers appears equally effective, but there is considerable merit in using a pyrE mutant as the host as, through the use of allele-coupled exchange (ACE) vectors, mutants created (by whatever means) can be rapidly complemented concomitant with restoration of the pyrE allele. This avoids the phenotypic effects frequently observed with high-copy-number plasmids and dispenses with the need to add antibiotic to ensure plasmid retention.",
keywords = "Journal Article",
author = "Muhammad Ehsaan and Sarah Kuehne and Minton, {Nigel P}",
year = "2016",
month = aug,
day = "10",
doi = "10.1007/978-1-4939-6361-4_4",
language = "English",
volume = "1476",
pages = "35--52",
journal = "Methods in molecular biology",
issn = "1064-3745",
publisher = "Springer",

}

RIS

TY - JOUR

T1 - Clostridium difficile Genome Editing Using pyrE Alleles

AU - Ehsaan, Muhammad

AU - Kuehne, Sarah

AU - Minton, Nigel P

PY - 2016/8/10

Y1 - 2016/8/10

N2 - Precise manipulation (in-frame deletions and substitutions) of the Clostridium difficile genome is possible through a two-stage process of single-crossover integration and subsequent isolation of double-crossover excision events using replication-defective plasmids that carry a counterselection marker. Use of a codA (cytosine deaminase) or pyrE (orotate phosphoribosyltransferase) as counter selection markers appears equally effective, but there is considerable merit in using a pyrE mutant as the host as, through the use of allele-coupled exchange (ACE) vectors, mutants created (by whatever means) can be rapidly complemented concomitant with restoration of the pyrE allele. This avoids the phenotypic effects frequently observed with high-copy-number plasmids and dispenses with the need to add antibiotic to ensure plasmid retention.

AB - Precise manipulation (in-frame deletions and substitutions) of the Clostridium difficile genome is possible through a two-stage process of single-crossover integration and subsequent isolation of double-crossover excision events using replication-defective plasmids that carry a counterselection marker. Use of a codA (cytosine deaminase) or pyrE (orotate phosphoribosyltransferase) as counter selection markers appears equally effective, but there is considerable merit in using a pyrE mutant as the host as, through the use of allele-coupled exchange (ACE) vectors, mutants created (by whatever means) can be rapidly complemented concomitant with restoration of the pyrE allele. This avoids the phenotypic effects frequently observed with high-copy-number plasmids and dispenses with the need to add antibiotic to ensure plasmid retention.

KW - Journal Article

U2 - 10.1007/978-1-4939-6361-4_4

DO - 10.1007/978-1-4939-6361-4_4

M3 - Article

C2 - 27507332

VL - 1476

SP - 35

EP - 52

JO - Methods in molecular biology

JF - Methods in molecular biology

SN - 1064-3745

ER -