2.2 Current Recombineering Techniiues 17on the overexpression of phage proteins that prepare and protect the DNA sub-
strate, have been developed to more efficiently induce the incorporation of
desired DNA segments into cells. Two popular methods are the Rec E/T and the
λ-Red prophage systems.
2.2.1 Recombineering Systems
The Rec E/T system encodes for two proteins, namely, RecE, a 5′ → 3 ′ exonucle-
ase, and RecT, an ssDNA binding protein [7]. The λ-Red system encodes for
three proteins: Exo (homologous to RecE), Beta (homologous to RecT), and
Gam, an inhibitor of the endogenous RecBCD exonuclease, which acts to protect
the foreign DNA from active degradation [12]. The foreign DNA to be recom-
bineered into the host genome may be in one of two forms depending on its
source. Synthetic oligonucleotides (oligos) will usually be ssDNA, while poly-
merase chain reaction (PCR)-amplified segments are double stranded. In order
to be incorporated into the host genome, the recombineering substrate includes
the desired DNA sequence to be incorporated and homology regions that flank
both sides of this DNA sequence. The homology regions direct the DNA sub-
strate to a specific location in the genome, where the endogenous replication
machinery uses it as a template for replication. Here, we will focus on the λ-Red
system, in which the Exo, Beta, and Gam proteins work in concert to induce
homologous recombination of DNA fragments into the host genome.
Currently, the most popular vectors for the λ-Red system are either the pKD46
or the pSIM5 plasmids [11, 13, 14]. Protein expression from these vectors is
induced by incubation with arabinose or at 42 °C, respectively. Both are addition-
ally temperature sensitive at 37 °C, which allows for plasmid curing following
expression of the λ-Red proteins. The standard λ-Red recombineering workflow
includes transforming the host strain with the recombineering plasmid of choice,
induction of the recombineering machinery, and additional transformation with
the desired recombineering substrate, followed by selection/screening for suc-
cessful recombinant strains [11].
2.2.2 Current Model of Recombination
Several models of the exact recombination mechanism exist; however, the “rep-
lication fork annealing model” is currently the most supported experimentally
(Figure 2.1). According to this model, if the recombineering substrate consists of
dsDNA, the λ-Red Exo protein, through its exonuclease activity, transforms it
into ssDNA [16]. This model suggests that although some dsDNA is being com-
pletely degraded by Exo molecules that digest the recombineering substrate from
both sides, in some cases one strand is digested completely before the other side
is attacked by another Exo molecule, rendering the resulting ssDNA immune
from further Exo digestion. If the recombineering substrate is ssDNA, no action
by Exo is required. In both cases, the (resulting) ssDNA strand is protected from
further degradation by endogenous nucleases via Beta, which binds to the ssDNA
and escorts it to single-stranded areas in the chromosome [17, 18]. Single-
stranded regions occur during DNA repair, transcription-induced supercoiling,