Biology Reference
In-Depth Information
These advances have allowed researchers to visualise several proteins simulta-
neously within the same live cell. In this section, we describe some of the consider-
ations needed when undertaking fluorescent protein labelling experiments and
highlight some of the more successfully utilised molecular systems used to label
proteins in
B. subtilis
,
E. coli
,
S. aureus
and
A. baylyi
.
2.1
Bacillus subtilis
B. subtilis
is one of the more highly utilised organisms used to study fluorescent pro-
tein localisation. There are numerous integrative plasmid systems available that can
give rise to either N- or C-terminal fusions, and they can be designed to integrate into
the chromosome at the target gene locus via a single-crossover integration, or to inte-
grate at a different locus via a double-crossover integration. Examples of some pop-
ular
B. subtilis
vectors are shown in
Figure 4.1
and
Table 4.1
. Single-crossover
integration involves the entire plasmid integrating into the chromosome and results
in the expression of C-terminal fluorescent protein fusions being driven by the target
gene's natural promoter, as shown schematically in
Figure 4.2
. This gives rise to a
fusion product with wild-type expression levels, which not only allows highly accu-
rate subcellular localisation data to be determined but also facilitates rapid quanti-
fication of protein abundance (
Doherty
et al.
, 2010; Buescher
et al.
, 2012
). These
plasmids typically contain auxiliary promoters such as IPTG or xylose-inducible
promoters to drive expression of genes downstream of the target gene in the event
they are located within an operon.
The
amyE
locus is a common choice for double-crossover integration in
B. subtilis
.
Integration vectors targeting this locus contain both 5
0
-and3
0
-regions of homology to
amyE
and are designed to express the gene fusion from either an IPTG or a xylose-
inducible promoter. A schematic illustrating a double-crossover integration into the
chromosome is shown in
Figure 4.2
. Unlike single-crossover vectors that arewell suited
for the production of C-terminal fusions, double-crossover vectors can also be used for
the generation of either N- or C-terminal fluorescent protein fusions (
Figure 4.1
;
Lewis
andMarston, 1999; Feucht and Lewis, 2001
). Integration via double crossover at
amyE
canbescreenedbyinactivationofthe
amyE
gene as outlined below. This type of system
is useful in the event the protein fusion is partially functional. For example, the partially
functional fusions to the essential cell division protein FtsZ expressed from the
amyE
locus can still be used to give accurate localisation data while it is functionally comple-
mented by the wild-type copy of the gene (
Feucht and Lewis, 2001
).
Numerous examples of co-localisation of two proteins exist in
B
.
subtilis
using
CFP/YFP or GFP/mCherry combinations (
Lewis
et al.
, 2000; Doherty
et al.
, 2010
).
Because integrative plasmids rely on regions of homology to integrate efficiently
into the chromosome, homology between their plasmid backbones can complicate
strain construction. Consequently, care needs to be taken when designing such
dual-labelling experiments. An effective plasmid combination for dual-labelling
experiments that avoids this problem is that of the
gfpmut3
containing pYG1 and
the
mCherry
containing pNG621 (
Figure 4.1
and
Table 4.1
;
Doherty
et al.
, 2010
).
These two plasmids share little sequence homology, which reduces the chance of
