PMC

A and B) Model for oligonucleotide-mediated mutagenesis. A linearized plasmid vector (left) is repaired by targeted recombination by S. cerevisiae yielding either a deletion (A) or a deletion and an insertion (B). In A) one oligonuclotide is used to target recombination and in B) two oligomers are used. C) Agarose gel of diagnostic PCR amplicons from a lacZ gene that is intact (WT) or has been mutated using a single oligonuclotide (Δ997) or two oligonucleotides (Δ9/496 and Δ500/496). The size of the targeted deletion is indicated, but with two primers is the sum of the numbers with a 30 bp insertion, i.e. a 966 bp change in amplicon size for Δ500/496.

The I-SceI meganuclease sequence (light blue box) was introduced into pMQ118 by oligonuclotide directed recombination generating pMQ236 (as in ). A. DNA sequences flanking the pigP homolog from S. marcescens were amplified with primers that direct recombination with each other and with pMQ236. These amplicons were introduced into S. cerevisiae with linearized pMQ236, generating a seamless pigP-Δ mutagenesis construct (C). This pMQ236 + pig-Δ construct was introduced into wild-type (WT) S. marcescens to generate a merodiploid, and this strain was transformed with an I-SceI expressing plasmid. D. The result of this allelic replacement strategy is restoration of the WT gene (left), or deletion of the pigP gene (right). E. Two colony color-types resulted from this allelic replacement strategy, one type was pink and the other was indistinguishable from the WT parental strain. F. PCR analysis indicates that the pink strains have the pigP-Δ, whereas the red strains retained the full-length pigP gene. These results support the model that PigP is a positive regulator of red pigment production in both S. marcescens and Serratia sp. ATCC 39006.