Use of the Red- Recombineering Method for Genetic Engineering - Genetic Engineering: Recent Developments in Applications - page 22

as follows. About 10 clones from 10 6 transformants obtained after electroporation
of P. ananatis SC17 strain by pRSFRedTER, were of larger size after being plated
on LB-agar with Cm. In LB-broth, bacteria from the “large” clones had a growth
rate similar to the control strain with the pRSFsacB plasmid, and induction of the
λ Red genes by IPTG caused only slight retardation in the growth of these cells.
Several of the selected "large" clones were cured from the plasmid on LB-agar
containing sucrose, and re-transformed with pRSFRedTER. All clones grown
after this re-transformation were of large size, similar to the parental clones.
Three of the pRSFRedTER transformants that grew well were used as recipient
strains for λ Red-mediated disruption of hisD gene. A PCR substrate, contain-
ing (attL λ -KmR-attR λ )-marker flanked by 40-bp homologous to the hisD gene,
was electroporated into these strains. From 100 to 150 KmRHis- clones per 108
survivors following electroporation were obtained for each tested recipient strain.
The insertion of the marker in the desired point of hisD gene was confirmed
by PCR-analysis of 10 independent KmRHis- clones in each case. The observed
integration frequency was similar to that obtained in the corresponding experi-
ments with E. coli [4-6]. One of the plasmid-less strain used as a recipient in this
experiment was named as SC17(0), and the obtained hisD strain constructed on
its basis - as SC17(0)hisD::(attL λ -KmR-attR λ ).
We tried to determine the nature of the mutation/mutations that provide re-
sistance to concerted expression of λ Red genes to P. ananatis. There were no
auxotrophic properties for SC17(0) strain growing on the M9 minimal media,
supplemented with different carbon sources, in comparison with initial SC17
strain [1]. One of the possible explanations for the SC17(0) resistance is the re-
duced level of accumulation of λ Red proteins in this strain. To test the level of
accumulation of λ Red proteins in SC17 and SC17(0), we performed SDS-PAGE
of extracts of both strains carrying pRSFRedTER plasmid. Unfortunately, bands
of λ Red proteins were not detected among the total cellular proteins even in
conditions of IPTG induction for the both plasmid-carrier strains. The reduction
in level of accumulation of λ Red proteins in SC17(0) strain, also, can be caused
by decreased copy-number of RSF1010-replicon carrying plasmids in this strain.
However, no reliable change in copy-number of pRSFsacB plasmid extracted from
the cells of P. ananatis SC17 or SC17(0) strains could be experimentally found.
On the other hand, the toxicity of the expression of λ Red genes has been detected
for the plasmid-carrier cells of SC17 strain grown even without IPTG addition
to the medium. In this case the transcription of the operon mediated by auto-
regulated PlacUV5-lacI genetic element has to be tightly repressed. According
to Skorokhodova et al. addition of 1 mM IPTG to the culture medium provides
an increase of transcriptional level up to 10-20 fold [31]. But, the transcription
level of λ Red genes under such conditions is not toxic for SC17(0). Hence, it is