Excisionase (Xis) proteins (LR clonase), derived from elements used during the bacterio-
phagellife cycle. DNA fragments flanked by recombination sites can be mixed in vitro
with vectors that also contain recombination sites, allowing the exchange of DNA frag-
ments and the generation of recombinant DNA. Such an approach avoids many of the dif-
ficulties associated with conventional cloning (inconvenient restriction sites, time-
consuming reactions, etc.). For Gateway cloning, theattsites have been modified so that
the orientation of the DNA fragments can be maintained during the excision and integration
process. Catalyzed by BP clonase, anattB1site specifically recombines with anattP1site to
produce anattL1site, while anattB2site specifically recombines with anattP2site to
produce anattL2site (Fig. 7.12). This allows PCR fragments flanked byattB1andattB2
sites to be inserted into pDONR vectors containing the reciprocalattPsites, thereby gen-
erating “entry clones” in which the chosen DNA fragments are flanked byattL1and
attL2sites.
Entry clones should be sequence-validated to provide a library of well-characterized
DNA fragments for insertion into “destination vectors.” Catalyzed by LR clonase, DNA
fragments flanked byattL1andattL2sites are then transferred, by a second recombination
Figure 7.12.A gene or promoter is amplified by PCR using DNA target-specific primers that contain
theattBsites (attB1andattB2) at the 5^0 and 3^0 ends, respectively. The purified PCR product, flanked
byattBsites, is mixed with a pDONOR vector that contains the correspondingattPsites. To this DNA
mix is added BP clonase enzyme (containing Int and IHF proteins). After 1 h incubation at 25 8 C, pro-
teinase K is added and incubated for a further 20 min at 37 8 C. This mix is used to transformE. coli
bacteria and plated on the appropriate antibiotic (in this example kanamycin) selecting transformants
containing the appropriate pENTRY clone.
7.3. GREATER DEMANDS LEAD TO INNOVATION 173