Use of the Red- Recombineering Method for Genetic Engineering - Genetic Engineering: Recent Developments in Applications - page 25

transfer of Marked Mutations by electroporation of
chromosomal dnA
General transduction is the most efficient and popular method for transfer of mu-
tations between different E. coli strains. Although P. ananatis and E. coli are close
relatives, the known E. coli transducing phages cannot infect P. ananatis cells.
Therefore, development of another method for transfer of mutations between P.
ananatis strains was necessary.
The electroporation of genomic DNA has been described for the transfer of ge-
netic markers between different backgrounds of E. coli and Pseudomonas [37,38].
We tried to apply this technique to P. ananatis. The SC17(0)hisD::(attL λ -KmR-
attR λ ) strain was used as a donor of KmR marker. Wild type strain SC17 was
used as a recipient for electro-transformation of chromosomal DNA.
Previously, it was shown that special electroporation conditions are needed for
the transformation of E. coli cells with large DNA molecules (see [39] for details).
Different cultivation conditions of recipient strain and parameters of electropora-
tion (electric field strength - E, time constant - τ ) were tested. For P. ananatis, the
highest yield of integrants (about 100 KmR His- integrants per 108 survivors fol-
lowing electroporation) was obtained under the following conditions. Recipient
strain was grown up to absorbance of 0.8 - 1.0. Then electro-competent cells were
prepared using 10 ml of culture as described in the "Plasmid electro-transforma-
tion" section (see "Methods"). Electroporation was performed at E = 12.5 kV/cm
and τ = 10 msec (resistance of 400 Ω and capacity of 25 µF). Electro-transforma-
tion with chromosomal DNA is very fast method: all procedures, including DNA
isolation and electroporation, can be performed in one day.
We found that marked chromosomal modification, obtained in P. ananatis
SC17(0) strain via λ Red-recombineering method, could be easily transferred
into the wild type SC17 strain by electroporation of chromosomal DNA. At the
time of writing up to ten different mutations had been combined in the chromo-
some of the SC17 strain by repeated electro-transformation with chromosomal
DNA followed by λ Int/Xis-driven excision of selective marker. The frequency of
marked mutation transfer varied from several up to several hundreds of integrants
per trial.
two-step λ red-Mediated introduction of unmarked
Mutations into P. ananatis chromosome
A two-step λ Red-mediated procedure for the introduction of unmarked mu-
tations was elaborated for P. ananatis SC17(0). It comprises: 1) λ Red-driven