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unlikely, that the synthesis of λ Red proteins in SC17 under the repressed condi-
tions could be significantly higher than in SC17(0) after induction.
Perhaps, mutation/mutations that are present in the genome of the SC17(0)
strain does not affect most of the genes encoding factors of global cellular regula-
tion. At least, the patterns of total cellular proteins separated by 2D-PAGE in
such a fashion that about 350 of individual polypeptides could be quantitatively
analyzed [33], were not different for the SC17 and SC17(0) strains (data not
shown).
Thus, the molecular mechanism of the resistance of the mutant SC17(0) strain
to concerted expression of all λ Red genes is unknown as yet. Nevertheless, as will
be shown below, various λ Red-driven modifications of bacterial chromosome
could be provided on the basis of this selected strain.
Use of the Combined λ Red-Int/Xis System for Introduction of
Multiple Modifications
Earlier, a λ Int/Xis-driven system for removing the markers from E. coli chro-
mosome was constructed, similar to one developed by Peredelchuk & Bennet
[34,35]. It comprised of the plasmids carrying removable markers of KmR or
CmR flanked by attL λ and attR λ sites and the plasmid pMW-intxis-ts with ther-
mo-sensitive pSC101-like replicon. This plasmid provided thermo-inducible ex-
pression of the xis-int genes under the control of λ P R promoter regulated by the
temperature sensitive λ CIts857 repressor. Even being partially induced at 37°C,
this system provided a high frequency (about 30%) of marker excision in E. coli.
The high frequency of marker eviction at +37°C was very important for use of the
system in P. ananatis because this bacterium has a lower temperature optimum
than E. coli and cannot grow at 42°C (the standard temperature for temperature
sensitive λ CIts857 repressor inactivation).
The pMW-intxis-ts plasmid contains ApR gene as a selective marker that was
not practicable for P. ananatis because of its high natural resistance to Ap. For this
reason, we have substituted this marker with the CmR gene. The resulting plas-
mid (pMW-intxis-cat) was introduced to the SC17(0)hisD::(attL λ -KmR-attR λ )
strain described above by electroporation. More than 30% of the transformants
grown at 37°C on the plates containing LB-agar with Cm had lost the Km resis-
tance. Loss of the KmR cassette in the kanamycin-sensitive colonies was verified
by PCR. Thus, the KmR cassette can be used in the next step of the chromosomal
modifications of this strain (Fig. 1A). Curing of the KmS clones from the pMW-
intxis-cat plasmid (with a frequency of 10%) was performed by re-streaking bac-
teria on LB-agar without antibiotic followed by cultivation at 37°C.
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