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FIG. 2. Schematic representation of the steps involved in gene replacement by double homologous recombination (homogenotization [107, 277]). The green lines represent regions targeted for disruption. (A) An antibiotic resistance gene (in this case, encoding a ß-lactamase that confers resistance to carbenicillin) has been inserted into the targeted gene that has been cloned into an IncP
plasmid (containing a kanamycin resistance gene [kan] in its backbone) and introduced into Agrobacterium. Double homologous recombination is allowed to take place. (B) Following double homologous recombination, the disrupted gene is exchanged onto the Ti plasmid (pTi). (C) A plasmid of the same incompatibility group as the first plasmid is introduced into Agrobacterium. An example is the IncP plasmid pPH1JI, containing a gentamicin resistance gene (gent). (D) Because plasmids of the same incompatibility group (in this case IncP) cannot replicate independently in the cell at the same time, selection for gentamicin resistance results in eviction of the other IncP plasmid, onto which has been exchanged the wild-type gene.
