BACKGROUND Escherichia coli strain EL350 contains chromosomally integrated phage lambda Red recombinase genes enabling this strain to be used for modifying the sequence of resident clones via recombineering. BAC and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2) copies per cell, the DNA is difficult to isolate in high yield and pu...

Generation of multiple genomic alterations is currently a time consuming process. Here, a method was established that enables highly efficient and simultaneous deletion of multiple genes in Escherichia coli. A temperature sensitive plasmid containing arabinose inducible lambda Red recombineering genes and a rhamnose inducible flippase recombinase was constructed to facilitate fast marker-free d...

We report the identification and functional analysis of nine genes from Gram-positive and Gram-negative bacteria and their phages that are similar to lambda (lambda) bet or Escherichia coli recT. Beta and RecT are single-strand DNA annealing proteins, referred to here as recombinases. Each of the nine other genes when expressed in E. coli carries out oligonucleotide-mediated recombination. To o...

The lambda phage Red recombination system can mediate efficient homologous recombination in Escherichia coli, which is the basis of the DNA engineering technique termed recombineering. Red mediated insertion of DNA requires DNA replication, involves a single-stranded DNA intermediate and is more efficient on the lagging strand of the replication fork. Lagging strand recombination has also been ...

"Recombineering," in vivo genetic engineering with short DNA homologies, is changing how constructs are made. The methods are simple, precise, efficient, rapid, and inexpensive. Complicated genetic constructs that can be difficult or even impossible to make with in vitro genetic engineering can be created in days with recombineering. DNA molecules that are too large to manipulate with classical...

We have developed an effective, easy-to-use two-step system for the site-directed insertion of large genetic constructs into arbitrary positions in the Escherichia coli chromosome. The system uses lambda-Red mediated recombineering accompanied by the introduction of double-strand DNA breaks in the chromosome and a donor plasmid bearing the desired insertion fragment. Our method, in contrast to ...

We have constructed a set of plasmids that can be used to express recombineering functions in some gram-negative bacteria, thereby facilitating in vivo genetic manipulations. These plasmids include an origin of replication and a segment of the bacteriophage lambda genome comprising the red genes (exo, bet and gam) under their native control. These constructs do not require the anti-termination ...

In this report, we describe the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecET proteins encoded by the lambda and Rac bacteriophages of Escherichia coli. The abil...