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Besides general cloning techniques we are mainly making use of the lambda Red/ET recombination system. The Red/ET technology and design of user-defined homology regions allows that any position of a target DNA molecule can be altered without having to rely on restriction endonucleases. The Red/ET system to engineer DNA molecules has initially been described by Francis A. Stewart and co-workers (Zhang et al., 1998).

Principle of Red/ET recombination. In recombineering, E. coli hosts are used to exercise homologous recombination between selected and user-defined target molecules. Redα is a 5’-3’ exonuclease, and Redβ is a single stranded DNA annealing protein that act consecutively after occurrence of a DNA double stranded break. Redα recesses the 5’ ends that will be bound by Redβ. During the induced double strand break repair process recombination occurs through user-defined homology regions shared by the two partner molecules. The schematic picture is adapted from a product manual of GeneBridges. 

In the recombination reaction, the Lambda Red protein RecE/Redα allows single strand degradation at chosen homologous regions, which is subsequently followed by single strand DNA binding of RecT/Redβ, facilitating pairing of homologous regions (see Figure). The actual homologous recombination occurs during DNA replication, and homologous exchange occurs via Holliday junction resolution. Definition of the homologous regions (usually between 50 and 60bp) decides over retrieval (e.g. from BAC to plasmid or from plasmid to plasmid backbone) or insertion (any given cassette into BAC or plasmid backbone).

The choice of homology arms defines the reaction direction. By choice of homology arms, either DNA fragments can be inserted into existing constructs (left) or fragments from large-scale genomic constructs can be retrieved into plasmid backbones (right). All reactions make use of the E. coli double strand DNA-break repair-pathway and are mediated by Red/ET components. Insertion and retrieval are driven by the respective replicating unit (depending on the ORI of either BAC or plasmid).