Biomedical Engineering Reference
In-Depth Information
Acc
I
Acc
I
Pvu
II
Pvu
II
Pvu
II
Acc
I
Pvu
II
Pvu
II
Sal
I
Pvu
II
tetR
Pvu
II
Sma
I
Xma
I
lacZ'
lacZ'
tetA
Nde
I
cat
pJB3Tc20
7069 bp
Pvu
II
Nde
I
pJB3Cm6
6227 bp
trfA
oriT
trfA
oriT
Fig. 8.4
Map and construction of
general-purpose broad-host-range
cloning vectors derived from plasmid
RP4. The restriction sites in the
polylinker downstream of the
lacZ
promoter are marked (t), and the sites
are, in the counterclockwise direction,
Hin
dIII,
Sph
I,
Pst
I,
Sal
I/
Hinc
II/
Acc
I,
Xba
I,
Bam
HI,
Xma
I/
Sma
I,
Kpn
I,
Sac
I and
Eco
RI. Sites in the polylinker that are
not unique are indicated elsewhere
on each vector. Note that the sites for
Nde
I and
Sfi
I are unique for all of the
vectors except pJB321.
Pneo
, promoter
from the neomycin resistance gene;
bla, kan, tet
and
cat
, genes encoding
ampicillin, kanamycin, tetracycline
and chloramphenicol resistance,
respectively. (Figure modified from
Blatny
et al
. 1997.)
Acc
I
Sfi
I
Acc
I
bla
Pneo
Sfi
I
oriV
bla
Pneo
Pvu
II
oriV
Pvu
II
Acc
I
Sal
I
Pst
I
Bg
III
Acc
I
Pvu
II
Sma
I
Hin
d
III
Pvu
II
Nde
I
Pvu
II
Xma
I
Pvu
II
lacZ'
lacZ'
kan
parDE
Pst
I
Acc
I
Sal
I
Nde
I
Acc
I
oriT
Nde
I
pJB3Km1
6052 bp
pJB321
5594 bp
trfA
oriT
Acc
I
trfA
bla
Sfi
I
bla
Sfi
I
Pneo
oriV
Pneo
oriV
Pvu
II
Pvu
II
used as vectors because their large size makes
manipulations difficult.
A number of groups have developed mini-IncP
plasmids as vectors and good examples are those
of Blatny
et al
. (1997). Their vectors are only 4.8 -
7.1 kb in size but can still be maintained in a wide
range of Gram-negative bacteria. All the vectors
share a common polylinker and
lacZ
oped by the inclusion of controllable promoters.
Representative examples of these vectors are shown
in Fig. 8.4.
An alternative way of using P-group plasmids as
cloning vectors has been described by Kok
et al
.
(1994). Their method combines the advantages
of high-copy-number pBR322 vectors with the con-
venience of conjugative plasmids. This is achieved
by converting the pBR322 vector into a transpos-
able element. Most pBR322 derivatives contain the
β
region, thereby
simplifying cloning procedures and identification of
inserts by blue/white screening (see p. 35) and most
carry two antibiotic-resistance determinants. All the
vectors retain the
oriT
(origin of transfer) locus,
enabling them to be conjugally transferred in those
cases where the recipient cannot be successfully
transformed or electroporated. Two other features
of these vectors deserve mention. First, the
parDE
region from the parent plasmid has been included in
some of the vectors, since this greatly enhances their
segregative stability in certain hosts. Secondly, the
trfA
locus on the vectors contains unique sites for
the restriction enzymes
Nde
I and
Sfi
I. Removal of
the
Nde
I-
Sfi
I fragment results in an increased copy
number. Expression vectors have also been devel-
′
-lactamase gene and one of two 38 bp inverted
repeats of transposon Tn2. By adding a second
inverted repeat, a transposable element is created
(Fig. 8.5). All that is missing is transposase activity
and this is provided by another plasmid, which is a
pSC101 derivative carrying the
tnpA
gene. To use
this system, the desired DNA sequence is cloned into
the transposition vector. The recombinant molecules
are transformed into an
E. coli
strain carrying the
P-group plasmid (e.g. R751) and the pSC101
tnpA
derivative and selection is made for the desired char-
acteristics. Once a suitable transformant has been
selected, it is conjugated with other Gram-negative
