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

Marinelli, LJ and Piuri, M and Swigoňová, Z and Balachandran, A and Oldfield, LM and van Kessel, JC and Hatfull, GF (2008) BRED: A simple and powerful tool for constructing mutant and recombinant bacteriophage genomes. PLoS ONE, 3 (12).

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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. © 2008 Marinelli et al.


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Details

Item Type: Article
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Marinelli, LJ
Piuri, M
Swigoňová, Zzus3@pitt.eduZUS30000-0002-6658-1392
Balachandran, A
Oldfield, LM
van Kessel, JC
Hatfull, GFgfh@pitt.eduGFH
Contributors:
ContributionContributors NameEmailPitt UsernameORCID
EditorBaker, Scott E.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Date: 17 December 2008
Date Type: Publication
Journal or Publication Title: PLoS ONE
Volume: 3
Number: 12
DOI or Unique Handle: 10.1371/journal.pone.0003957
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Refereed: Yes
PubMed Central ID: PMC2597740
PubMed ID: 19088849
Date Deposited: 24 Jul 2012 18:53
Last Modified: 02 Feb 2019 15:58
URI: http://d-scholarship.pitt.edu/id/eprint/13097

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