Simple generation of site-directed point mutations in the Escherichia coli chromosome using Red (R)/ET (R) recombination

Background: Introducing 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. Results:
Here 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 (R)/ET (R)
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.
Conclusion: Chromosomal modifications performed by rpsL
counter-selection may also be used for other bacteria that contain
an rpsL homologue, since Red (R)/ET (R) Recombination has been
applied to several enteric bacteria before.