Use of the Red- Recombineering Method for Genetic Engineering - Genetic Engineering: Recent Developments in Applications - page 20

ssDNA greater than 35 nucleotides in length and mediates pairing the one with a
complementary target [15-17].
he λ Red-mediated recombineering technology developed initially for modi-
fication of the genome of Escherichia coli K12 [2,4-7], was later broadened to
other E. coli strains including enteropathogenic ones [18], and to Salmonella
[19,20], Shigella [21,22], Yersinia [23,24], Pseudomonas [25], as well. Probably,
one of the factors impeding application of this system in other hosts is the toxicity
of expression of the λ Red genes for the cells.
In the present study, to overcome this toxicity for Pantoea ananatis, the special
strain resistant to simultaneous expression of the λ Red genes was selected. Using
this mutant, construction of all types of chromosomal rearrangements previously
obtained in E. coli by recombineering was reproduced. The approach described
may be used for adjusting the technology to other hosts.
results
Construction of the New Broad-Host-Range λ Red-Expressing
Plasmid
To provide regulated expression of the λ Red genes in different bacteria, the
plasmid pRSFRedTER [GenBank:FJ347161] based on the broad-host-range
replicon of RSF1010 [26] has been constructed. This plasmid is useful for λ
Red-mediated recombineering because: 1) the replicon is stably maintained
in many Gram-negative [27] and some Gram-positive bacteria [28]; 2) the λ
Red genes are placed under the control of the P lacUV5 promoter recognized by
different bacterial RNA polymerases [29,30]; 3) the auto-regulated element
P lacUV5 -lacI provides IPTG-inducible expression of the λ Red genes with low
basal level [31]; 4) the plasmid contains the levansucrase gene from B. subtilis
allowing rapid and efficient recovery of this plasmid from the cells in a me-
dium containing sucrose.
To test recombineering efficiency, pRSFRedTER mediated disruption of galK
gene in E. coli MG1655 chromosome by integration of the PCR-generated DNA
fragment carrying the KmR gene from pUC4K flanked by attL λ and attR λ sites
(attL λ -KmR -attR λ ) was performed. The constructed plasmid provided about
300 transformants per 108 survivors following electroporation. A similar frequen-
cy was obtained when pKD46 plasmid [4] was used as λ Red-expressing plasmid.
In each case a chromosome structure of ten KmR colonies was confirmed with
PCR analysis.