Biomedical Engineering Reference
In-Depth Information
without tandem duplication of the chromosomal
segments. In this case, the plasmid DNA is linearized
before transformation, as shown in Fig. 8.2. The
same technique can be used to generate deletions.
The gene of interest is cloned, a portion of the gene
replaced
in vitro
with a fragment bearing an anti-
biotic marker and the linearized plasmid trans-
formed into
B. subtilis
, with selection made for
antibiotic resistance.
Bg
/
II
Bg
/
II
B
C
AD
Pst
I
Cut with
Pst
I
and transform
with selection for Km
R
Cloning in Gram-negative bacteria
other than
E. coli
To clone DNA in non-enteric bacteria, a plasmid
cloning vehicle is required which can replicate in the
selected organism(s). Under normal circumstances,
E. coli
will be used as an intermediate host for trans-
formation of the ligation mix and screening for
recombinant plasmids. Therefore, the vector must
be able to replicate in
E. coli
as well. The options
which are available are to generate a shuttle vector
or to use a broad-host-range plasmid as a vector. If
a small plasmid can be isolated from the bacterium
of interest, then it is easy to splice it into an existing
E. coli
vector to generate a shuttle vector. Recent
examples of this approach are the construction
of vectors for use in
Pasteurella
(Bills
et al
. 1993),
Desulfovibrio
(Rousset
et al
. 1998) and
Thermus
(De
Grado
et al
. 1999). This approach is particularly
useful if the selectable markers used in
E. coli
also function in the new host. Then one can take
advantage of the many different specialist vectors
(see Chapter 5) which already exist, e.g. expres-
sion vectors, secretion vectors, etc. If the selectable
markers are not expressed in the new host, then
extensive manipulations may be necessary just to
enable transformants to be detected.
With broad-host-range plasmids, there is a high
probability that the selectable markers will be ex-
pressed in the new host and confirming that this is
indeed the case is easy to do. However, the naturally
occurring plasmids do not fulfil all the criteria for an
ideal vector, which are:
• small size;
• having multiple selectable markers;
• having unique sites for a large number of restric-
tion enzymes, preferably in genes with readily
scorable phenotypes.
A
A
B
B
C
C
D
D
Plasmid
Pst
I
Bg
/
II
Pst
I
Chromosome
Fig. 8.2
Insertion of plasmid DNA into the chromosome by
a double crossover event. The
B. subtilis
DNA is shown in
grey and the letters A to D represent different chromosomal
sequences. Vector DNA is shown in white and other
vector-borne genes in pink.
This technique is particularly useful if one wishes
to construct a recombinant carrying a single copy of
a foreign gene.
Once a recombinant plasmid has integrated into
the chromosome, it is relatively easy to clone adja-
cent sequences. Suppose, for example, that a vector
carrying
B. subtilis
DNA in the
Bam
HI site (Fig. 8.1)
has recombined into the chromosome. If the recom-
binant plasmid has no
Bgl
II sites, it can be recovered
by digesting the chromosomal DNA with
Bgl
II, ligat-
ing the resulting fragments and transforming
E. coli
to Ap
R
. However, the plasmid which is isolated will
be larger than the original one, because DNA flank-
ing the site of insertion will also have been cloned.
In this way, Niaudet
et al
. (1982) used a plasmid
carrying a portion of the
B. subtilis ilvA
gene to clone
the adjacent
thyA
gene.
Genes cloned into a plasmid and flanked by regions
homologous to the chromosome can also integrate
