Fig. 2.

Creation of rescue constructs by recombineering. At the top left is shown a vector used to retrieve sequences from the bacterial artificial chromosome (BAC) shown at the top right. Some key features of the vector are marked. These include the two ends (5′P and 3′P) of a P transposable element ¹⁶ and a pair of “homology arms”, ~500 bp segments (marked A and B) whose sequences come from the two ends of a region of interest (marked r.o.i.) within the BAC. Upon introduction by electroporation of a BamHI-linearized vector into a BAC-containing strain that has been engineered to overexpress recombination proteins, gap repair by homologous recombination promotes the transfer to the vector of the entire segment of the BAC that lies between the arms. Two attractive features of this recombineering (recombination-mediated genetic engineering) procedure ⁴² is that it does not depend on fortuitously placed restriction sites (any sequence from the BAC can be amplified and cloned as a homology arm) and that it employs the natural replication machinery of E. coli to achieve the transfer of genetic information and thus is much less prone to introduction of errors than polymerase chain reaction-based methods.