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Bacteriophages, the historic model organisms facilitating the initiation of molecular biology, are still important candidates of numerous useful or promising biotechnological applications. Development of generally applicable, simple and rapid techniques for their genetic engineering is therefore a validated goal. In this article, we report the use of bacteriophage recombineering with electroporated DNA (BRED), for the first time in a coliphage. With the help of BRED, we removed a copy of mobile element IS1, shown to be active, from the genome of P1vir, a coliphage frequently used in genome engineering procedures. The engineered, IS-free coliphage, P1virdeltaIS, displayed normal plaque morphology, phage titre, burst size and capacity for generalized transduction. When performing head-to-head competition experiments, P1vir could not outperform P1virdeltaIS, further indicating that the specific copy of IS1 plays no direct role in lytic replication. Overall, P1virdeltaIS provides a genome engineering vehicle free of IS contamination, and BRED is likely to serve as a generally applicable tool for engineering bacteriophage genomes in a wide range of taxa., (© 2011 The Authors. Microbial Biotechnology © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

E. coli K-12, being one of the best understood and thoroughly analyzed organisms, is the workhorse of genetic, biochemical, and systems biology research, as well as the platform of choice for numerous biotechnological applications. Genome minimization/remodeling is now a feasible approach to further enhance its beneficial characteristics for practical applications. Two genome engineering techniques, a lambda Red-mediated deletion method and a suicide (conditionally replicative) plasmid-based allele replacement procedure are presented here. These techniques utilize homologous recombination, and allow the rapid introduction of virtually any modifications in the genome.