Biology Reference
In-Depth Information
electro-competent cells were prepared as described above. From 200 to 500 ng
of a PCR-generated linear dsDNA or 100 ng of a ss-oligos were used for trans-
formation. Electroporation was carried out at electric field strength of 25 kV/cm
and time constant of 5 msec for both types of DNA substrates. The chromosome
structure of the obtained transformants was verified in PCR with galK-t1/galK-
t2 primers for E. coli galK gene disruption, hisD-t1/hisD-t2 for P. ananatis hisD
gene disruption and for insertion of the double selective/contra-selective marker
into the P. ananatis hisD.
Gene disruption provided with pKD46 plasmid was performed as described
in (4).
P. Ananatis electro-transformation with chromosomal dnA
Cells were grown in LB medium up to OD
600
= 0.8-1.0. Electro-competent cells
were prepared as described above. From 1 to 2 mg of a chromosomal DNA, iso-
lated using a Genomic DNA Isolation Kit (Sigma), was used for transformation.
Electroporation was carried out with an electric field strength of 12.5 kV/cm and
time constant of 10 msec.
sds-PAGe
SDS-PAGE of cell extracts was performed according to Laemmli [59] with poly-
acrylamide gel with linear gradient of concentrations from 10% to 15%.
Abbreviations
Ap: ampicillin; Ap
R
: ampicillin resistance; Cm: chloramphenicol; Cm
R
: chloram-
phenicol resistance; cat: chloramphenicol resistance gene; Km: kanamycin; Km
R
:
kanamycin resistance; kan: kanamycin resistance gene; sacB: gene encoding the
levansucrase; attL
λ
: attL site of phage
λ
; attR
λ
: attR site of phage
λ
; attB
λ
: attB site
of phage
λ
; IPTG: isopropyl-
β
-D-thiogalactopyranoside; nt: nucleotide; bp: base
pair(s); oligos: oligonucleotides; PCR: polymerase chain reaction; ssDNA: single-
stranded DNA; dsDNA: double-stranded DNA.
Authors' contributions
JIK and YH are the project leaders in AGRI and in Ajinomoto, respectively. JIK
obtained SC17(0), designed the main experiments concerned with
λ
Red-driven
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