Bacteriophage recombineering in the lytic state using the lambda red recombinases

Abstract

SummaryBacteriophages, 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.

Klebsiella Phage ΦK64-1 Encodes Multiple Depolymerases for Multiple Host Capsular Types

“…One technique, which uses phage lambda as a model system, involves phage infection, competent cell preparation, and electroporation of recombineering DNA substrates (28,29). The second technique is bacteriophage recombineering of electroporated DNA (BRED), which was first described for mycobacteriophage engineering (30) and subsequently applied to construction of coliphage mutants (31). In BRED, bacterial cells inducibly expressing recombination functions are electroporated with a combination of phage DNA template and a targeting substrate.…”

“…Cycling conditions were as follows: 96°C for 3 min, followed by 30 cycles of 96°C for 30 s, 54°C for 15 s, and 72°C for 2 min. The amplified products were ligated to ApaLI-digested pKD46, a plasmid that encodes components of the -Red recombinase system (31). The resulting plasmid was electroporated into the hosts of ⌽K64-1 (e.g., K1 Klebsiella strains) and selected on LB agar supplemented with trimethoprim at 75 g/ml at 30°C.…”

Genetically Engineered Phages: a Review of Advances over the Last Decade

“…2) (63,66). This technique was first applied by Marinelli et al to modify mycobacteriophages (66) and has since been expanded to modify phages that target bacterial hosts other than mycobacteria for which recombineering systems are available, such as Escherichia coli and Salmonella enterica (67,68). BRED can be used to delete, insert, and replace genes, as well as to create point mutations in phage genomes.…”

Comparison of CRISPR and marker based methods for the engineering of phage T7

“…An alternative to positive selection is to reduce the number of phage that have to be screened to find the desired mutant by increasing the efficiency of homologous recombination. An example of this is the BRED system (bacteriophage recombineering with electroporated DNA) that has been used to genetically modify mycobacteriophages and coliphages [19,20]. BRED exploits the mycobacterial recombineering system, in which expression of the RecE/RecT-like proteins of the mycobacteriophage Che9c confers high levels of homologous recombination and facilitates simple allelic exchange using a linear DNA substrate.…”

“…Another method to reduce the number of phage that require screening is the use of a Yeast Artificial Chromosome (YAC) in Saccharomyces cerevisiae [19,20]. Here, a phage genome can be cloned into a YAC, either in one piece or as PCR fragments assembled through gap-repair cloning [23].…”