Format

Send to:

Choose Destination
See comment in PubMed Commons below
PLoS One. 2008;3(12):e3957. doi: 10.1371/journal.pone.0003957. Epub 2008 Dec 17.

BRED: a simple and powerful tool for constructing mutant and recombinant bacteriophage genomes.

Author information

  • 1Pittsburgh Bacteriophage Institute and Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

Abstract

Advances in DNA sequencing technology have facilitated the determination of hundreds of complete genome sequences both for bacteria and their bacteriophages. Some of these bacteria have well-developed and facile genetic systems for constructing mutants to determine gene function, and recombineering is a particularly effective tool. However, generally applicable methods for constructing defined mutants of bacteriophages are poorly developed, in part because of the inability to use selectable markers such as drug resistance genes during viral lytic growth. Here we describe a method for simple and effective directed mutagenesis of bacteriophage genomes using Bacteriophage Recombineering of Electroporated DNA (BRED), in which a highly efficient recombineering system is utilized directly on electroporated phage DNA; no selection is required and mutants can be readily detected by PCR. We describe the use of BRED to construct unmarked gene deletions, in-frame internal deletions, base substitutions, precise gene replacements, and the addition of gene tags.

PMID:
19088849
[PubMed - indexed for MEDLINE]
PMCID:
PMC2597740
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Public Library of Science Icon for PubMed Central
    Loading ...
    Write to the Help Desk