Use of the Red- Recombineering Method for Genetic Engineering - Genetic Engineering: Recent Developments in Applications - page 32

construction of the Linear dsdnA Fragment to exchange
native smai recognition site in the P. Ananatis hisd Gene by
Xhoi site
First, his-XhoI-1 and his-XhoI-2 oligos complementary to each other were an-
nealed. Both oligos contained sequences corresponding to the XhoI restriction
site in the center and arms homologous to the sequence surrounding native SmaI
site of the P. ananatis hisD. As a result, the short dsDNA fragment containing the
XhoI recognition site in its center and 33 bp long arms homologous to the target
region, was obtained. The obtained fragment was amplified and extended by PCR
with the primers his-SL and his-SR. The resultant DNA fragment generated by
PCR contained XhoI recognition site in its center flanked with 82 bp arms ho-
mologous to the appropriate site in P. ananatis hisD gene.
Plasmid electro-transformation
An overnight culture of P. ananatis strain grown at 34°C with aeration was di-
luted with fresh LB broth 100 times and the cultivation was continued up to
the OD 600 = 0.5-0.8. Cells from ten milliliters were washed three times with an
equal volume of deionized ice water followed by washing with 1 ml of 10% cold
glycerol and resuspended in 35 µ l of 10% cold glycerol. Just before electropora-
tion, 10-100 ng of the plasmid DNA dissolved in 2 µ l of deionized water was
added to the cell suspension. The procedure of plasmid electro-transformation
was performed using the GenePulser and Pulse Controller ("BioRad", USA). The
applied pulse parameters were: electric field strength of 20 kV/cm, time constant
of 5 msec. After electroporation, 1 ml of LB medium enriched with glucose (5
g/l) was immediately added to the cell suspension. Then the cells were cultivated
under aeration at 34°C for 2 h and plated on LB-agar containing the appropriate
antibiotic. This was followed by an overnight incubation at 34°C. A competence
of P. ananatis cells, determined for RSF1010 plasmid, was 106 CFU per µ g of
DNA. Typically, 105-106 antibiotic resistant colonies were obtained per 108 sur-
vivors following electroporation.
Gene rearrangement
Overnight cultures of P. ananatis or E. coli strains harbouring the plasmid,
expressing appropriate λ Red genes, grown in LB broth with Cm (for pRS-
FRedTER, pRSFGamBet plasmids) or Km (for pRSFRedkan, pRSFGamBetkan
plasmids) were diluted 100 times with the same fresh medium supplemented with
1 mM IPTG for induction of the λ Red genes. At culture density of 0.5-0.6,