Simple generation of site-directed point mutations in the Escherichia coli chromosome using Red®/ET® Recombination

Abstract

BackgroundIntroducing point mutations into bacterial chromosomes is important for further progress in studies relying on functional genomics, systems- and synthetic biology, and for metabolic engineering. For many investigations, chromosomal systems are required rather than artificial plasmid based systems.ResultsHere we describe the introduction of a single point mutation into the Escherichia coli chromosome by site-directed mutagenesis without leaving any selection marker. We used Red®/ET® Recombination in combination with rpsL counter-selection to introduce a single point mutation into the E. coli MG1655 genome, one of the widely used bacterial model strains in systems biology. The method we present is rapid and highly efficient. Since single-stranded synthetic oligonucleotides can be used for recombination, any chromosomal modification can be designed.ConclusionChromosomal modifications performed by rpsL counter-selection may also be used for other bacteria that contain an rpsL homologue, since Red®/ET® Recombination has been applied to several enteric bacteria before.

Identification of a Target Gene and Activating Stimulus for the YpdA/YpdB Histidine Kinase/Response Regulator System in Escherichia coli

“…E. coli strains and their genotypes are listed in Table 1. Mutants were constructed with the E. coli Quick and Easy gene deletion kit (Gene Bridges) and the Bac modification kit (Gene Bridges), as reported previously (17). Both kits rely on the Red/ET recombination technique.…”

Identification of ArgP and Lrp as Transcriptional Regulators of lysP, the Gene Encoding the Specific Lysine Permease of Escherichia coli

“…E. coli JM109 (60) was used as a carrier for all plasmids. For the construction of the reporter strains, MG-LR and MG-CR, a method based on rpsL counterselection in combination with the Red/ET recombination system was employed (19) according to the protocol recommended by the technical manual of the Quick and Easy E. coli deletion kit (Gene Bridges). Briefly, the coding sequence of the target gene (lysP or cadBA) was replaced by an rpsL-neo cassette (Gene Bridges) by Red/ET recombination in strain MG16R (Table 1) to give strains MG16R4 and MG16R12.…”

Site-Directed Mutagenesis Using Oligonucleotide-Based Recombineering

“…A cassette containing an rpsLneo fusion gene was used. Clones exhibited Km R and Sm S (Heermann et al, 2008). The desired point mutation for the gene of interest had already been introduced into one of the two 50-bp homology arms of the rpsL-neo cassette.…”

“…Recombinants that deleted the wt rpsL could be selected by their resistance to streptomycin. After successful recombination, the wt copy of rpsL was removed, and the gene of interest was re-established, now containing the desired mutation (Heermann et al, 2008).…”

“…It has been applied to Escherichia coli, Salmonella, and a range of other Gram-negative bacteria, as well as to bacteriophages, cosmids and bacterial artificial chromosomes (BACs). It was demonstrated that single-stranded DNA (ssDNA) oligonucleotides can be used as substrates for recombineering in E. coli , Heermann et al, 2008 and BACs (Swaminathan et al, 2001). However, most commonly linear, double-stranded DNA (dsDNA) has been used as the targeting construct (Maresca et al, 2010), e.g., for chromosomal gene replacement (Murphy, 1998), whole gene disruption (Datsenko et al, 2000) or the development of novel cloning strategies, including subcloning of BAC DNA ).…”