Fig. 9. Recombineering-mediated mutagenesis. (A) `Blind' mutagenesis. To perform a site-specific change in a fragment within a target plasmid, a PCR fragment or oligonucleotide that contains the desired mutation is transformed into bacteria that contain recombineering functions and a target plasmid. Recombination results in the incorporation of the desired mutation: substitution, deletion or insertion. The bacteria are then screened by PCR for the proper mutagenic event. (B) Mutagenesis using a positive/negative selectable marker. First, in the positive-selection step, a PCR fragment containing a positive (+)/negative (-) selectable marker is transformed into bacteria that contain recombineering functions and the target plasmid. Individual colonies containing the correct recombinant plasmid are then selected. Second, during the counterselection step, a PCR fragment containing the desired change, such as a tag, might replace the positive/negative selectable marker. Counterselection or negative selection may result in the selection of a correct recombinant plasmid that can be identified through PCR. (C) RecA-assisted modification. A specialized plasmid that contains a selectable marker (+), a counterselectable marker (-), RecA and a mutation flanked by two homology boxes (A and B) is transformed into bacteria. During a first recombination event, identified through selection, this plasmid can integrate through homology box A (shown) or B (not shown), resulting in a co-integrate. During a second recombination event, identified through counterselection, this co-integrate can resolve to the original plasmid (not shown) or the modified plasmid (shown).