Biology Reference
In-Depth Information
assay (
Bisicchia
et al.
, 2010
). Briefly, transformants are stabbed onto plates contain-
ing 1% starch (for integrations at
amyE
) or 0.4% lichenan (for integrations at
bglS
)
and onto replica plates without any addition and grown overnight. These plates are
then flooded with iodine (0.1% in 1N HCl) or Congo red dye (0.4%). Colonies with
reduced or without halos will have an integration at these loci. Stocks should be made
from the corresponding colony on the replica plate. The half-life of both CFP and
YFP in
B. subtilis
is approximately 2 h, enabling expression profiles of promoter
fusions with these two reporter proteins to be compared (
Bisicchia
et al.
, 2010
). This
vector suite has several advantages and offers considerable versatility for fusion gen-
eration: (1) fusions are present in single copy on the chromosome; (2) the activity of
promoters from other bacteria can be investigated in
B. subtilis
and (3) CFP and YFP
are spectrally distinct so that the activities of two different promoters can be
examined in the same strain. This is further facilitated by the use of two different
integration sites (
amyE
and
bglS
) and selection for resistance to two different anti-
biotics (kanamycin and chloramphenicol). This suite of vectors is available at the
ECE225, pCFP(
bglS
); ECE226, pYFP(
bglS
); ECE227, pGFP(
amyE
); ECE228,
pCFP(
amyE
) and ECE229, pYFP(
amyE
).
7.2.3
pFSB79
This vector is designed to generate transcriptional
gfp
fusions. The
gfp
รพ
gene is
utilized on this plasmid. It encodes a fast-folding GFP variant with enhanced fluo-
rescence (
Scholz
et al.
, 2000
) although the stability of this protein in
B. subtilis
has
not been established. Promoter fusions are integrated into the
amyE
locus of the
B. subtilis
chromosome with selection for chloramphenicol (
Brigulla,
et al.
, 2003
).
7.2.4
pAH321
This vector is designed to generate transcriptional fusions using the
luxABCDE
system from
P. luminescens
(
Schmalisch
et al.
, 2010
). Promoter-containing
fragments must have
Eco
RI-
Sal
I ends for directional cloning into the
Eco
RI-
Sal
I-digested pAH321 plasmid (
McLoon
et al.
,2011
). The ribosome binding sites
for all the
lux
genes have been optimized for expression in
B. subtilis
and the linear-
ized recombinant plasmid is integrated into the neutral
sac
locus of the chromosome
with selection for chloramphenicol resistance (
McLoon
et al.
, 2011
).
7.3
High-throughput cloning and plasmid preparation
Promoter fusions can easily be generated manually in batches of up to 24 constructs
or robotically using high-throughput procedures. The LIC procedure outlined above
lends itself well to automation and high-throughput application because sets of for-
ward primers have common 5
0
extensions, as do the sets of reverse primers (
Au
et al.
,
2006; Fogg and Wilkinson, 2008
). A list of target promoter sequences is established
and compatible sets of PCR primer sequences with user-specified properties are gen-
erated by computer. Oligonucleotides are obtained from commercial suppliers in
