Figure 2.

Construction of the unmarked nucleotide exchange in the P. ananatis hisD gene. A) Construction of a dsDNA fragment with appropriate mutation in the center. First, his-XhoI-1 and his-XhoI-2 oligos are annealed to each other. The nucleotide exchange of interest included in the sequence of his-XhoI-1/his-Xho2 olig is indicated by asterisks. The resulting dsDNA fragment is then used as DNA-template for PCR-amplification with his-SL and his-SR oligos. As a result a linear dsDNA fragment, harboring a XhoI restriction site and 82 bp long arms homologous to the target region of hisD gene, was obtained. B) The cassette containing dual selective/contra-selective marker is integrated into the target point of hisD gene. The constructed in vitro linear dsDNA fragment or ssDNA harboring appropriate mutation in the center is then integrated into the chromosome of this strain by the λ Red recombination system. As a result, the dual selective/contra-selective marker is eliminated from the chromosome with simultaneous introduction of the desired mutation into the hisD gene. This mutation leads to substitution of the native SmaI restriction site by the XhoI restriction site and restoration of the amino-acid sequence of HisD protein. Integrants are selected as colonies resistant to sucrose. Such colonies are subsequently tested for Cm sensitivity, ability for growth without histidine and presence of XhoI restriction site in the target chromosome point.