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May 15, 2009 (Vol. 29, No. 10)

New Tool Recombineers the DNA of E. coli

Red/ET Recombination Technology Opens Opportunities in Industrial Biotech

  • DNA Modification

    Click Image To Enlarge +
    Figure 1. The central step in Red/ET Recombineering is the exchange of genetic information between two DNA fragments containing homology regions.

    While many systems for genetic engineering in E. coli allow only for modifications at specific positions (e.g., transposons, group II introns), the Red/ET system is sequence independent. Red/ET recombination is based either on the phage proteins RecE/RecT from Rac prophage or Reda/Redb from phage l and requires short flanking homologous stretches of only 50 bp (Figure 1).

    Homology arms, therefore, of any given sequence can easily be introduced to a linear fragment by PCR. The required phage genes are provided by an expression plasmid that can be transferred into any E. coli strain, and a temperature-sensitive replication origin allows for a subsequent removal of the plasmid.

  • Click Image To Enlarge +
    Figure 2. Application of suitable linear DNA fragments allows modifications such as gene knockout through disruption (A) or deletion (B) of an open reading frame, insertion of a foreign gene (C) or change of a promoter (D).

    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).

  • Click Image To Enlarge +
    Figure 3. Red/ET allows every type of DNA engineering possible regardless of target size or type of modification.

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