Use of the Red- Recombineering Method for Genetic Engineering - Genetic Engineering: Recent Developments in Applications - page 21

Another plasmid carrying λ Red genes and named pRSFRedkan
[GenBank:FJ347162], has been constructed via substitution of CmR and B. sub-
tilis sacB genes by the KmR gene from pUC4K [32].
Concerted Expression of λ Red Genes is Highly Toxic for the
P. Ananatis Wild-Type Cells
Clones of P. ananatis SC17 strain [1] obtained after electroporation by pRS-
FRedTER and plated on a solid LB-medium supplemented with Cm (50 µ g/ml)
were of very small size. Bacteria from these colonies grew very slowly in compari-
son with bacteria carrying pRSFsacB plasmid, which served as a vector for cloning
of λ Red genes. Addition of IPTG (1 mM) for induction of expression of λ Red
genes, led to complete cessation of the growth of pRSFRedTER containing cells.
This effect was not detected for the cells carrying pRSFsacB and was based on the
toxicity of the expression of λ Red genes.
To establish which component of the λ Red system caused the toxic effect,
we constructed the pRSFGamBet [GenBank;FJ347163] plasmid lacking the exo
gene encoding 5' → 3' exonuclease.
It is well known that exonuclease activity is necessary for integration of dsD-
NAs only; integration of ssDNAs, such as chemically synthesized oligos, requires
only recombinase (product of the bet gene, see [8]). To test the functional activity
of the constructed pRSFGamBet, the plasmid was used to promote recombina-
tion between the artificial ss-oligos comprising two 36-nt homologies to the galK
sequences and chromosome of the E. coli MG1655galK::(attL λ -KmR-attR λ )
strain. As a result of recombination a native structure of galK gene was restored.
Between (1.5-2.5) × 104 Gal+ integrants per 108 survivors following electropora-
tion were obtained in three independent experiments.
When introduced into P. ananatis SC17 strain, the pRSFGamBet plasmid
did not inhibit cell growth even under the induced conditions (in the presence
of 1 mM IPTG). Hence, the detected toxicity of pRSFRedTER was apparently
caused by exo expression, or by simultaneous expression of all λ Red genes in P.
ananatis cells.
Selection of the Recipient Strain for λ Red-Mediated
Recombineering in P. Ananatis
A mutant P. ananatis strain, SC17(0), resistant to concerted expression of all λ
Red genes, and thus manifesting the properties of a suitable recipient strain for λ
Red-mediated integration of the linear DNAs into the chromosome, was obtained