Recombineering

Abstract

Recombineering, a recently developed technique for efficient genetic manipulation of bacteria, is facilitated by phage-derived recombination proteins and has the advantage of using DNA substrates with short regions of homology. This system was first developed in E. coli but has since been adapted for use in other bacteria. It is now widely used in a number of different systems for a variety of purposes, and the construction of chromosomal gene knockouts, deletions, insertions, point mutations, as well as in vivo cloning, mutagenesis of bacterial artificial chromosomes and phasmids, and the construction of genomic libraries has been reported. However, these methods also can be effectively applied to the genetic modification of bacteriophage genomes, in both their prophage and lytically growing states. The ever-growing collection of fully sequenced bacteriophages raises more questions than they answer, including the unknown functions of vast numbers of genes with no known homologs and of unknown function. Recombineering of phage genomes is central to addressing these questions, enabling the simple construction of mutants, determination of gene essentiality, and elucidation of gene function. In turn, advances in our understanding of phage genomics should present similar recombineering tools for dissecting a multitude of other genetically naïve bacterial systems.

A standardized workflow for surveying recombinases expands bacterial genome‐editing capabilities

“…Fortunately, recombineering is evolving to enable large‐scale genomic editing with a host of applications, ranging from increases in genomic diversity to the optimization of metabolic pathways for biosynthesis (Wang et al ., 2009; Gallagher et al ., 2014). Recombineering refers to directed genetic recombination between foreign DNA and endogenous homologies during DNA replication, as mediated by phage‐derived recombinases (Yu et al ., 2000; Ellis et al ., 2001; Marinelli et al ., 2012). Modern recombineering technologies originated in Escherichia coli , in which the β, Exo and γ proteins of λ phage were first exploited in tandem to introduce double‐stranded (ds) DNA into the lagging strand of transient replication forks (Murphy, 1998; Yu et al ., 2000).…”

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).…”

“…The phage lysate is subsequently recovered and checked for incorporation of the desired DNA (70). The yield of recombinant phages obtained by using this technique is about 0.5 to 2%, which is higher than the yield obtained by homologous recombination but still low, so screening for the mutant phages remains challenging (63,66,70). This technique can potentially be adapted to other phages and other bacterial species by introducing the Red system via plasmids (without the rest of the phage), or another recombination machinery, into host bacteria that can be targeted by the phage to be engineered.…”

“…Without an efficient phage screening method, finding the desired clone is labor-intensive at best. Therefore, a reporter gene, usually encoding luciferase or a fluorescent protein, is commonly cloned along with the gene of interest to facilitate the identification of mutant phages by detecting the reporter (59,(62)(63)(64)(65). Because the recombination rates obtained with this technique are low, it is improbable that targeting multiple loci at the same time will result in an organism carrying all the desired mutations.…”

“…BRED can be used to delete, insert, and replace genes, as well as to create point mutations in phage genomes. It consists of coelectroporating the recombineering substrates, i.e., phage DNA and double-stranded DNA (dsDNA), into electrocompetent bacterial cells carrying a plasmid that encodes proteins promoting high levels of homologous recombination, such as the RecE/RecT-like proteins (63,66). The dsDNA substrate comprises the DNA fragment to be inserted along with regions of homology to the loci immediately up-and downstream of the region of the phage genome to be modified (66,69).…”