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Figure 1. Scheme of a construction of multiple chromosomal modifications, using the combined λ Red-Int/Xis
system. A) Selective marker M flanked by attL λ /attR λ is used for introduction of an appropriate mutation into
the chromosome by λ Red-dependent recombination. Then the marker is eliminated from the chromosome by
λ Int/Xis site-specific recombination. As a result, only the 31 bp long attB λ sequence linked to the mutation
remains in the chromosome. Selective marker M can then be used in the next step of the introduction of
multiple chromosomal modifications. B) The sequence of the attB λ site. One of the six ORFs provided by this
sequence does not contain stop codons. Hence, it is possible to design an "in-frame" deletion of a gene. Asterisks
mark stop codons.
he attB λ site (31 bp in length), remaining in the chromosome after marker
excision, contains six possible reading frames. One of these reading frames does
not contain stop codons (Fig. 1B). Therefore, usage of the removable markers
flanked with attL λ and attR λ sites allows design and construction of "in frame"
deletions.
Using the λ Red-driven chromosomal modification followed by λ Int/Xis-me-
diated excision of the selective marker, it was possible to provide, step-by-step, the
multiple chromosomal modifications in P. ananatis SC17(0) strain. This approach
was repeatedly used for different modifications. Among them were 1) combina-
tions of the simple or "in frame" deletions of several genes/operons; 2) integra-
tion of the marked heterologous genes into the chromosome of P. ananatis and 3)
modification of the regulatory regions of the genes of interest [36]. Up to the pres-
ent, several P. ananatis strains carrying more than 10 different modifications have
been constructed using this strategy; the presence of multiple attB λ sites in their
chromosomes did not hamper the repeated exploitation of this system.
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