Biology Reference
In-Depth Information
normal locus and its control regions intact. Thus expression of each promoter fusion
is established in the wild-type genetic background. A high frequency of integrants
is ensured with a minimum of 400 bp of DNA homology. When integrating
promoter fusions at a neutral heterologous locus (e.g.
amyE
in
B. subtilis
)bya
double-crossover event, the promoter-containing DNA fragment can be of any
length, although the restrictions delimiting the 3
0
-end still apply. A LIC system is
particularly suited to high-throughput generation of promoter fusions using several
of the plasmids described in the following section (
Botella
et al.
, 2010
;
Bisicchia
et al.
, 2010
;
Figure 1.1
). LIC compatible sequences are designed into the 5
0
-ends
of the primers used to amplify the promoter-containing DNA fragments as shown
in
Figure 1.1
. Treatment of the fragments with T4 DNA polymerase in the presence
of dTTP generates single-stranded ends that are compatible with similarly treated
ends of plasmid pBaSysBioII (see below). This cloning methodology has several
advantages including its suitability for automated high-throughout cloning proce-
dures, a high transformation frequency, no background (false-positive) transformants
and directional cloning of the insert so that the promoter always reads into the
reporter gene.
7.2
Plasmids
The following briefly describes some plasmids used to generate transcriptional
fusions that allow comparative analysis of promoter activities. Several plasmids
designed specifically for use in
B. subtilis
are available from the
Bacillus
Genetic
Stock Centre (the BGSC ID number is given in parenthesis).
7.2.1
pBaSysBioII
Plasmid pBaSysBioII (ECE223) is designed to integrate promoter fusions into the
B. subtilis
chromosome by a single-crossover event at homologous loci
(
Figure 1.1
). Plasmid pBaSysBio II encodes a pBR322 replicon and ampicillin resis-
tance gene allowing constructs to be cloned in
E. coli
(
Botella
et al.
, 2010
). It also
encodes a promoterless
gfpmut3
gene isolated from pJBA27 (
Bo Andersen
et al.
,
1998
), with an optimized ribosome binding site (AAGGAGG) located 7 bp upstream
of the ATG start codon, and a LIC cloning site. Digestion of the plasmid with
Sma
I,
followed by treatment with T4 DNA polymerase in the presence of dATP, generates
single-stranded ends that are compatible with the promoter-containing fragments
with LIC ends that have been treated with T4 DNA polymerase in the presence of
dTTP (
Figure 1.1
). Annealed plasmid and insert can be transformed directly into
E. coli
without ligation.
7.2.2
pGFPamy, pCFPamy, pYFPamy/pGFPbglS, pCFPbglS, pYFPbglS
vector suite
A series of plasmids has been constructed to expand the methodology for generating
promoter fusions (
Bisicchia
et al.
, 2010
). The
gfpmut3
(from pBaSysBioII),
yfp
(from pIYFP,
Veening
et al.
, 2004
) and
cfp
(from pDR200,
Doan
et al.
, 2005
)
