Use of the Red- Recombineering Method for Genetic Engineering - Genetic Engineering: Recent Developments in Applications - page 27

This procedure could be applied for introduction of unmarked mutations
to the genome of SC17(0) strain. Transfer of the obtained unmarked muta-
tions to other strains by electro-transformation with chromosomal DNA is
difficult because of impossibility of direct selection of integrants. Hence, to
provide construction of unmarked mutations in the genome of the P. ananatis
strains, sensitive to expression of all λ Red genes, we performed the method
of λ Beta-driven integration of single-stranded oligos into the chromosome
of SC17 strain.
λ Beta-Driven Integration of Single-Stranded Oligos
As mentioned above, ssDNAs, e.g. oligos containing short flanks homologous
to the target site, can be integrated into the E. coli chromosome using only the
product of the λ bet gene [8]. As the expression of gam and bet genes from pRS-
FGamBet plasmid was not toxic towards P. ananatis SC17 even under induced
conditions, we tried to perform λ Beta-driven integration of oligos into the chro-
mosome of this strain.
The hisD::PlacUV5-sacB-cat chromosomal modification was transferred into
the SC17 strain by electro-transformation with genomic DNA isolated from
SC17(0)hisD::PlacUV5-sacB-cat strain. The resulting strain, selected on LB me-
dium supplemented with Cm, was named SC17hisD::PlacUV5-sacB-cat. The
pRSFGamBet-kan [GenBank:FJ347164] plasmid was constructed by substitu-
tion of sacB and cat genes of pRSFGamBet for the KmR gene from pUC4K. This
plasmid was introduced to the obtained SC17hisD::PlacUV5-sacB-cat strain. It is
known from the literature [8,42] that the efficiency of λ Red-dependent integra-
tion of the oligos depends on a direction of replication through the recombination
site. The direction of replication at the hisD locus in the P. ananatis chromosome
is not known. Hence, the plasmid-carrier cells were independently electroporated
with hisD-XhoI-1 and hisD-XhoI-2 ss-oligos complementary to each other. Cells
were plated on LB medium containing 30% sucrose. No colonies were observed
after 24-hour cultivation when hisD-XhoI-2 olig was used for electroporation.
About 200 clones were obtained after electro-transformation with hisD-XhoI-1
olig. To test the phenotype of the obtained transformants, 100 clones were rep-
licated on the following solid mediums: LB medium supplemented with 30%
sucrose, LB medium supplemented with Cm and M9 minimal medium. Seven
of the tested clones had SucRCmSHis+ phenotype. These colonies were further
verified by PCR and restriction of the amplified product. The presence of the
expected mutation (substitution of the SmaI by XhoI recognition site) was con-
firmed in all of the seven SucRCmSHis+ clones.