Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow Print PDF (56K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (13)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by El Karoui, M.
Right arrow Articles by Gruss, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by El Karoui, M.
Right arrow Articles by Gruss, A.
Related Collections
Right arrow Mutagenesis
Right arrow DNA transfer
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research, Vol 27, Issue 5 1296-1299, Copyright © 1999 by Oxford University Press

ARTICLES

Gene replacement with linear DNA in electroporated wild-type Escherichia coli

M El Karoui, SK Amundsen, P Dabert and A Gruss
Genetique Appliquee-URLGA, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas, France.

Gene replacement using linear double-stranded DNA fragments in wild- type Escherichia coli transformation is generally inefficient due to exonucleolytic degradation of incoming DNA. Recombination-proficient strains, in which the exonucleolytic activity of RecBCD is inactivated, have been used as transformation recipients to overcome this difficulty. Here we report that gene replacements using linear double- stranded donor DNA can be achieved in wild-type E.coli if electrocompetent cells are used. Using a plasmid target, we obtained 10(2)-10(3) gene replacement events/microgram linear DNA. Using an independent chromosomal target, approximately 60 gene replacement events/microgram linear DNA were obtained. The presence of Chi sites on the linear DNA, which are known to block DNA degradation and stimulate recombination in E.coli, had no effect on gene replacement efficiency in either case. RecBCD-mediated exonucleolytic activity was found to be diminished in electroporated cells. Electrotransformation thus provides a simple way to perform gene replacements in many E.coli strains.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Appl. Environ. Microbiol.Home page
I. R. Monk, C. G. M. Gahan, and C. Hill
Tools for Functional Postgenomic Analysis of Listeria monocytogenes
Appl. Envir. Microbiol., July 1, 2008; 74(13): 3921 - 3934.
[Abstract] [Full Text] [PDF]

Home page
 Appl. Environ. Microbiol.Home page
A. E. Bloor and R. M. Cranenburgh
An Efficient Method of Selectable Marker Gene Excision by Xer Recombination for Gene Replacement in Bacterial Chromosomes
Appl. Envir. Microbiol., April 1, 2006; 72(4): 2520 - 2525.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S. Uzzau, N. Figueroa-Bossi, S. Rubino, and L. Bossi
Epitope tagging of chromosomal genes in Salmonella
PNAS, December 6, 2001; (2001) 261348198.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
A. Quiberoni, I. Biswas, M. El Karoui, L. Rezaiki, P. Tailliez, and A. Gruss
In Vivo Evidence for Two Active Nuclease Motifs in the Double-Strand Break Repair Enzyme RexAB of Lactococcus lactis
J. Bacteriol., July 1, 2001; 183(13): 4071 - 4078.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
M. Orford, M. Nefedov, J. Vadolas, F. Zaibak, R. Williamson, and P. A. Ioannou
Engineering EGFP reporter constructs into a 200 kb human {beta}-globin BAC clone using GET Recombination
Nucleic Acids Res., September 15, 2000; 28(18): e84 - e84.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S. Uzzau, N. Figueroa-Bossi, S. Rubino, and L. Bossi
Epitope tagging of chromosomal genes in Salmonella
PNAS, December 18, 2001; 98(26): 15264 - 15269.
[Abstract] [Full Text] [PDF]


Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.