Use of the Red- Recombineering Method for Genetic Engineering - Genetic Engineering: Recent Developments in Applications - page 29

possesses reduced level of the synthesis of λ Red proteins. But this possibility can
not be completely rejected.
Nevertheless, even without information concerning the nature of the SC17(0)
mutation, it is possible to use the corresponding strain for the desired λ Red-
mediated rearrangements of P. ananatis chtomosome. All types of chromosome
modifications constructed in E. coli by the λ Red-mediated recombineering, have
been successfully reproduced in SC17(0) with pRSFRedTER as λ Red-expressing
plasmid. Typically, the yield of recombinants varied from several tens to several
hundreds per trial.
Using SC17(0) as the initial recipient for λ Red-promoting modifications it is
subsequently possible to transfer the marked mutation to other P. ananatis strains
of interest using the method of electro-transformation with chromosomal DNA.
This method is a unique way to combine the set of marked mutations constructed
in different P. ananatis strains into a single strain. In addition, potentially, co-
transfer of rather closed mutations could be prevented by digestion of the chro-
mosomal DNA by the appropriate restriction endonucleases, whose recognition
sites are located between the mutations.
As the number of combined mutations of interest exceeds the number of avail-
able antibiotic resistance markers, curing of the intermediate strains from the
used selective markers is necessary. Moreover, the presence of antibiotic resistance
genes in the genomes of the industrial strains is rigorously restricted by the legisla-
tions of different countries. A wide variety of systems for marker curing based on
the site-specific recombination systems (Cre/lox, Flp/FRT) are well-known [54-
57]. These systems provide "symmetrical" recombination reaction between two
identical sites flanking the removing marker, e.g. FRTxFRT = 2FRT. In this case,
after removing the marker, the active site remains in the chromosome. Repeated
action of such systems can lead to inversion or deletion of extended chromosomal
fragments caused by site-specific recombination between the sites remaining at
different points in the chromosome. Therefore, use of systems providing "asym-
metrical" recombination reaction would be preferable. One such system is the
site-specific recombination system of λ phage. It includes the Int and Xis proteins
encoded by int and xis genes that, together with the host factors (IHF, RecA and
Fis), provide the following reaction: attL λ x attR λ = attP λ +attB λ . Peredelchuk &
Bennet [34] were the first who used this system for removal of the selective mark-
ers flanked by attL λ and attR λ sites. The attB λ site remaining in the chromosome
after marker excision cannot recombine with attL λ or attR λ site in the next steps
of strain construction. Thus, repeated action of the system would not influence
the strain stability. Certainly, residual attB λ sites could recombine with each other
via host general recombination system or provoke replication errors, especially if
many left over scars (attB λ ) are presented in chromosome and their positions were