...
To use the λ red recombineering system to modify target DNA (orange), linear donor dsDNA (blue) (ssDNA usage is not covered here) that includes 5′ and 3′ "homology arms" (H1/H2) matching the target DNA is electroporated into E. coli expressing the λ red enzymes, usually encoded on a temporary plasmid. These enzymes then catalyze the homologous recombination of the donor DNA with the target DNA sequence. The homology arms (H1/H2) of the donor DNA must match the region immediately 5′ and 3′ to the target sequence to be inserted into. Sequence in between the homology arms of the donor DNA replaces the sequence between the matching homology sequences in the target DNA. A selection, counterselection, or screening gene marker is usually included to select or screen for recombinants, and these markers are often designed to be removed by a second round of recombineering coupled with counterselection of a counterselectable marker, or using site-specific recombinases like the Flp-FRT system which requires FRT sequences around the region to be removed by Flp recombinase expressed (green) from a second plasmid.
The example below shows how the E. coli "Keio" gene deletion collection was constructed, wherein each non-essential gene in E. coli was deleted while minimizing disruptions to any translationally-coupled genes inside operons by using homology arms that preserve the start codon of the gene B to be deleted and the Shine-Dalgarno sequence of the downstream gene C that is internal to gene B toward its end.
dsDNA Procedure
...