reaction, to pDEST vectors containingattR1andattR2sites. The resulting recombinant
DNA constructs are known as “expression clones.” Here, the recombination product of
theattL1andattR1sites is anattB1site and the recombination product of theattL2and
attR2sites is anattB2site (Fig. 7.13).
To select the correct recombination product for the BP and LR reactions, a combination
of positive and negative selectable markers are employed. Positive selection is afforded by
alternative antibiotic selection, while negative selection is afforded by theccdBgene, the
product of which inhibits the activity of DNA gyrase, leading ultimately to cell death.
E. coli bacteria transformed with vectors containing theccdBgene (i.e., pDONR or
pDEST vectors) or by cointegrate intermediates, cannot grow. Only bacteria containing
the desired recombinant construct that lacks theccdBgene and contains the appropriate
antibiotic resistance marker gene can survive (Figs. 7.12 and 7.13). The propagation of
pDONR vectors and pDEST vectors is achieved using theE. colistrain DB3.1, which con-
tains a mutant DNA gyrase, which is unaffected by theccdBgene product.
Figure 7.13.A gene or promoter contained within the pENTRY clone flanked byattLsites (attL1
andattL2) is mixed with a pDESTINATION vector that contains the correspondingattRsites. To
this DNA mix is added LR clonase enzyme (containing the Int, IHF, and Xis proteins). After 1 h incu-
bation at 25 8 C, proteinase K is added and incubated for a further 20 min at 37 8 C. This mix is used to
transformE. colibacteria and plated on the appropriate antibiotic (in this example spectinomycin)
selecting transformants containing the appropriate pEXPRESSION clone.
174 RECOMBINANT DNA, VECTOR DESIGN, AND CONSTRUCTION