Gene Cloning

286 Gene Cloning
sac B
Ab
Non-replicating
plasmid
Target gene
Chromosome
Single recombination event
Ab
sac B
Target gene
1 2
Target gene
Ab
Figure 10.3 Pop-in, pop-out in bacteria. The first recombination event leads
to insertion of the entire plasmid into the chromosome. Cells that undergo this
event will become antibiotic resistant. Selection against the presence of the
plasmid sequences (by growth on sucrose, which is lethal in the presence of the
sacB gene) will lead to colonies that have lost the plasmid sequence by a second
recombination event. Depending on which of the flanking sequences is involved in
this second event, it will either regenerate the original sequence (1) or lead to
the swapping of the target gene with the antibiotic resistance marker (2), which
is the desired outcome.
A key feature of this method is that the plasmid cannot replicate in the
organism into which it is introduced, and is hence referred to as a “suicide
vector”. The plasmid is transformed into the organism, and selection is
made for the resistance marker on the plasmid. As the plasmid cannot
replicate, the only way that the cells can become resistant to the marker
and give rise to a colony is for recombination to occur between the plasmid
and the chromosome. The most common event will be a single cross-over,
which as shown in Figure 10.3 will integrate the entire plasmid onto the
chromosome. Once this has happened, the target gene is still present. The
recombination event can now be reversed by selecting against the presence
of the plasmid, by growing the cells on sucrose. Cells that still have the