bred a simple and powerful tool for constructing mutant and recombinant bacteriophage genomes培育一个简单而强大的工具为构建突变和重组噬菌体的基因组.pdf

BRED: A Simple and Powerful Tool for Constructing Mutant and Recombinant Bacteriophage Genomes ˇ ´ Laura J. Marinelli, Mariana Piuri, Zuzana Swigonova, Amrita Balachandran, Lauren M. Oldfield, Julia C. van Kessel, Graham F. Hatfull* Pittsburgh Bacteriophage Institute and Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America 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. ˇ ´ Citation: Marinelli LJ, Piuri M, Swigonova Z, Balachandran A, Oldfield LM, et al. (2008) BRED: A Simple and Powerful Tool for Constructing Mutant and Recombinant Bacteriophage Genomes. PLoS ONE 3(12): e3957. do