Biomedical Engineering Reference
In-Depth Information
Table 8.2
B. subtilis
-
E. coli
shuttle plasmids.
Replicon
Markers
Size
Plasmid (kbp) E. coli B. subtilis E. coli B. subtilis
Comments
pHV14
4.6
pBR322
pC194
Ap, Cm
Cm
pBR322/pC194 fusion. Sites:
Pst
I,
Bam
HI,
Sal
I,
Nco
I
(Ehrlich 1978)
pHV15
4.6
pBR322
pC194
Ap, Cm
Cm
pHV14, reversed orientation of pC194 relative to pBR322
pHV33
4.6
pBR322
pC194
Ap, Tc, Cm
Cm
Revertant of pHV14 (Primrose & Ehrlich 1981)
pEB10
8.9
pBR322
pUB110
Ap, Km
Km
pBR322/pUB110 fusion (Bron
et al
. 1988)
pLB5
5.8
pBR322
pUB110
Ap, Cm, Km
Cm, Km
Deletion of pBR322/pUB110 fusion,
Cm
R
gene of pCl94
Segregationally unstable (Bron & Luxen 1985). Sites:
Bam
HI,
Eco
RI,
Bql
III (in
Km
R
gene),
Nco
I (in
Cm
R
gene)
pHP3
4.8
pBR322
pTA1060
Em, Cm
Em, Cm
Segregationally stable pTA1060 replicon (Peeters
et al
.
1988). Copy number
c
. 5. Sites:
Nco
I (
Cm
R
gene),
Bcl
I
and
Hpa
I (both
Em
R
gene)
pHP3Ff
5.3
pBR322
pTA1060
Em, Cm
Em, Cm
Like pHP3; phage f1 replication origin and packaging
signal
pGPA14
5.8
pBR322
pTA1060
Em
Em
Stable pTA1060 replicon. Copy number
c
. 5.
a
-Amylase-based selection vector for protein export
functions (Smith
et al
. 1987). MCS of M13
mp
11 in
lacZ
a
pGPB14
5.7
pBR322
pTA1060
Em
Em
As pGPA14, probe gene TEM-
b
-lactamase
pHP13
4.9
pBR322
pTA1060
Em, Cm
Em, Cm
Stable pTA1060 replicon. Copy number
c
. 5. Efficient
(shotgun) cloning vector (Haima
et al
. 1987). MCS of
M13
mp
9 in
lacZ
a
LacZ
a
not expressed in
B. subtilis
. Additional sites:
Bcl
I
and
Hpa
I (both
Em
R
gene)
pHV1431 10.9
pBR322
pAM
b
1
Ap, Tc, Cm
Cm
Efficient cloning vector based on segregationally stable
pAM
b
1 (Jannière
et al
. 1990). Copy number
c
. 200. Sites:
Bgm
HI,
Sal
I,
Pst
I,
Nco
I. Structurally unstable in
E. coli
pHV1432
8.8
pBR322
pAM
b
1
Ap, Tc, Cm
Cm
pHV1431 lacking stability fragment orfH. Structurally
stable in
E. coli
pHV1436
8.2
pBR322
pTB19
Ap, Tc, Cm
Cm
Low-copy-number cloning vector (Jannière
et al
. 1990)
Structurally stable
A major contributing factor to structural instabil-
ity of recombinant DNA in
B. subtilis
appears to be
the mode of replication of the plasmid vector (Gruss
& Ehrlich 1989, Jannière
et al
. 1990). All the
B. sub-
tilis
vectors described above replicate by a rolling-
circle mechanism (see Box 8.2). Nearly every step in
the process digresses or could digress from its usual
function, thus effecting rearrangements. Also, single-
stranded DNA is known to be a reactive intermediate
in every recombination process, and single-stranded
DNA is generated during rolling-circle replication.
If structural instability is a consequence of rolling-
circle replication, then vectors which replicate by
the alternative theta mechanism could be more
stable. Jannière
et al.
(1990) have studied two poten-
tially useful plasmids, pAM
1 and pTB19, which
are large (26.5 kb) natural plasmids derived from
Streptococcus (Enterococcus) faecalis
and
B. subtilis
,
β
