Use of the Red- Recombineering Method for Genetic Engineering - Genetic Engineering: Recent Developments in Applications - page 31

Table 1. Strains and plasmids used or generated in this study.
Media and Growth conditions
E. coli and P. ananatis strains were cultivated with aeration in LB medium at 37°C
and 34°C, respectively. The following antibiotic concentrations were used to se-
lect transformants and to maintain the plasmids: Km - 40 mg/l, Cm - 50 mg/l.
The M9 salt medium supplemented with galactose (1 g/l) or glucose (1 g/l) was
used to select Gal + or His + cells.
recombinant dnA techniques
DNA manipulations were performed according to standard methods [58]. Re-
strictases were provided by “Fermentas” (Lithuania). T4-DNA ligase was from
Promega (USA). All reactions were performed according to the manufacturer's
instructions. PCR was carried out with Taq-polymerase (“Fermentas”). Primers
were purchased from “Syntol” (Russia).
construction of integrative cassettes
To provide cassettes for λ Red-dependent integration, the appropriate selective
marker was amplified by PCR with oligos containing on their 5'-ends 36-nt se-
quences homologous to the target region. To disrupt E. coli galK and P. ananatis
hisD genes, a removable Km R marker flanked by attL λ and attR λ was amplified
with galK-5/galK-3 and hisD-5/hisD-3 primers, respectively. The pMW-attL λ -
Km R -attR λ plasmid was used as DNA template. To obtain insertion into the P.
ananatis hisD gene, the Plac-sacB-cat cassette was amplified in PCR with his-
Plac-5/his-cat-3 primers using pRSFPlacsacB plasmid as template.