Combineering

Combineering, short for combinatorial genetic engineering, is a molecular biology technique that employs homologous recombination in bacteria to introduce precise genetic modifications such as insertions, deletions, or site‑directed mutations into plasmid or chromosomal DNA. The core of the method is the use of single‑stranded DNA (ssDNA) or double‑stranded DNA (dsDNA) substrates that are engineered to contain sequences homologous to the target locus. These substrates are introduced into recombination‑competent bacterial hosts, usually Escherichia coli, where proteins of the RecA‑dependent λ Red recombination system (Exo, Beta, and Gam) facilitate the strand exchange and integration events.

The technique was pioneered in the late 1990s by José García‑Martínez and colleagues, who demonstrated efficient gene replacement in E. coli using PCR products containing short homology arms. Since then, specialized strains such as SW102 and SLOE have been engineered to enhance recombination efficiency and reduce unwanted recombination events. Combining the method with other tools—such as synthetic gene libraries, CRISPR/Cas systems, or in vivo selection markers—has expanded its utility to high‑throughput genome editing, metabolic pathway optimization, and the construction of mutation libraries for protein engineering.

Combineering offers several advantages over conventional plasmid-based cloning, notably the ability to edit genomes directly without the need for multiple restriction sites, a lower risk of off‑target effects compared to oligonucleotide‑based mutagenesis, and compatibility with large synthetic DNA constructs. Its simplicity, speed, and versatility have made it an essential technique in bacterial genetics, synthetic biology, and functional genomics research.