Use of the Red- Recombineering Method for Genetic Engineering - Genetic Engineering: Recent Developments in Applications - page 30

rather close to each other. Such events would lead to deletions of chromosomal
fragments.
We adjusted the λ Int/Xis-dependent system for use in high-efficient marker
excision in P. ananatis. Note that it is also possible, in particular, to design marker-
less strains, carrying "in-frame" deletions.
Finally, the two-step procedure for introducing the unmarked mutations into
the P. ananatis genome was demonstrated using B. subtilis sacB gene as a counter-
selective marker. Desirable mutants were achieved at the second stage via λ Red-
mediated integration of the short dsDNA in SC17(0) or ssDNA in any other P.
ananatis strain carrying the dual selective/counter-selective marker in the target
point of the genome.
Up to the present, the developed λ Red-mediated method has been used for
deletion of more than 50 P. ananatis genes whose products were involved in cen-
tral metabolism, respiration, transcription regulation, etc. Several marker-less P.
ananatis strains carrying multiple (> 10) different chromosomal modifications
including "in-frame" deletions, point mutations, rearrangements of regulatory
regions, in particular, were constructed for basic research and applied purposes
using combined application of the λ Red-Int/Xis systems and electro-transforma-
tion with chromosomal DNA.
conclusion
he λ Red-mediated recombineering has been adjusted for rapid and efficient
construction of genome rearrangements in P. ananatis. In combination with the
established procedures of λ Int/Xis-dependent marker elimination and electro-
transformation with chromosomal DNA, this method provides a simple route
to obtaining marker-less strains carrying multiple mutations of different types
(deletions, substitutions of regulatory regions, integration of heterologous genes,
and point mutations). The described approach of selection of the recipient strain
resistant to expression of λ Red genes could be useful in exploiting λ Red-recom-
bineering in other bacteria.
Methods
strains and Plasmids
Strains and plasmids used or generated in this study are listed in Table 1.