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What is recombineering?

The development of genetic engineering revolutionized modern biology, allowing direct manipulation of an organisms DNA. Key to this technology was the discovery of restriction enzymes, proteins that when purified, cut DNA at precise short sequences in vitro. Different DNA molecules, cut with the same restriction enzyme(s), could then be joined together by DNA ligase to generate a recombinant molecule. Such genetically engineered plasmid DNA was routinely transformed into Escherichia coli for further analysis and manipulation. Although these genetic engineering techniques are powerful and continue to be used today, a new highly efficient method for directly modifying DNA within E. coli and other bacteria has been developed in the last ~15 years: recombineering.

Recombineering is in vivo homologous recombination-mediated genetic engineering. The homologous recombination is mediated by bacteriophage-based recombination systems such as the λ Red system, RecET from the Rac prophage, or others. In contrast to classical in vitro genetic engineering, recombineering does not rely on restriction enzymes. Thus, the location of restriction sites is no longer an issue, and the user defines the construct and its location to the base pair. Like genetic engineering, recombineering can be used to make deletions, point mutations, duplications, inversions, fusions and tags. Briefly, recombineering is performed by introducing linear DNA substrates containing the desired change and short flanking homologies to the target DNA into cells expressing the phage-encoded recombination enzymes. These enzymes recombine the linear DNA at the target, yielding recombinant molecules.