Genome-borne selection markers that are flanked by recognition sequences of a sequence-specific recombinase (FLP or Cre)—so-called FRT or loxP sequences— can be removed in a rapid and safe way by short expression of the appropriate enzyme. Thus, the removal of the selection markers allows for the successive modification of several genes, leaving only a FRT or loxP scar behind (Figure 2). Based on this strategy, up to seven genes have been modified in client projects.
Red/ET recombination enables rapid, easy, and precise modification of the E. coli genome. Foreign DNA fragments can be selectively inserted into any desired position. This technology facilitates the development of customized production strains for already existing biotechnological products.
There is evidence to suggest this technique is also applicable to other microorganisms besides E. coli, which remains the organism most widely used by molecular biologists.
Furthermore, Red/ET Recombineering is not limited to chromosomal modifications. The ability to clone and subclone large DNA molecules presents a variety of new ways to simplify conventional DNA engineering exercises. With this method changes can be directed to a chosen DNA sequence and point mutations can be introduced at any specific site of a target DNA molecule like a BAC or cosmid regardless of its size (Figure 3).