Biomedical Engineering Reference
In-Depth Information
to a region of the chromosome, then the outcome
is quite different. The homologous region may be
excised and incorporated into the chromosome by
the normal recombination process, i.e. substitution
occurs via a double crossover event. If only a single
crossover occurs, the entire recombinant plasmid is
integrated into the chromosome.
It is possible to favour integration by transferring
a plasmid into a host in which it cannot replicate and
selecting for a plasmid-borne marker. For example,
Stoss
et al
. (1997) have constructed an integrative
vector by cloning a neomycin resistance gene and
part of the
amyE
(alpha amylase) gene of
B. subtilis
in
plasmid pBR322. When this plasmid is transferred
to
B. subtilis
, it is unable to replicate, but, if selection
is made for neomycin resistance, then integration
of the plasmid occurs at the
amyE
locus (Fig. 8.1).
Integration of recombinant DNA
When a recombinant plasmid is transferred to an
unrelated host cell, there are a number of possible
outcomes:
• It may be stably maintained as a plasmid.
• It may be lost.
• It may integrate into another replicon, usually the
chromosome.
• A gene on the plasmid may recombine with a
homologous gene elsewhere in the cell.
Under normal circumstances, a plasmid will be
maintained if it can replicate in the new host and will
be lost if it cannot. Plasmids which will be lost in
their new host are particularly useful for delivering
transposons (Saint
et al
. 1995, Maguin
et al
. 1996).
If the plasmid carries a cloned insert with homology
Ap
R
Bam Bam
Cm
R
Bg
/
II
Bg
/
II
Bg
/
II
Bg
/
II
Recombination
Bam
H
I
Bam
H
I
Bg
/
II
Cm
R
Ap
R
Bg
/
II
Bg
/
II
Removal by
Bg
/
II
digestion
followed by ligation
Bg
/
II
Bam
H
I
Bam
H
I
Fig. 8.1
Cloning DNA sequences
flanking the site of insertion. The red
bar on the plasmid represents a
Bam
HI
fragment of
B. subtilis
chromosomal
DNA carrying the
amyE
gene. Note that
the plasmid has no
Bgl
II sites. (See text
for details.)
Cm
R
Ap
R
