Characterization of the Reporter System in a Multi-Well Plate Reader:
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To characterize the DarR reporter system, the ''E. coli'' strain BL21 was transformed either with [[Part:BBa_K1045017|BBa_K1045017]] or with [[Part:BBa_K1045013|BBa_K1045013]] as a control. In [[Part:BBa_K1045013|BBa_K1045013]], ''gfp'' is placed downstream of a strong promoter and the DarR operator. This vector does not encode DarR. The strong fluorescence signal of cells transformed with [[Part:BBa_K1045013|BBa_K1045013]] indicated that GFP was produced. However, when transformed with [[Part:BBa_K1045017|BBa_K1045017]] ('''Fig. 1'''), the bacteria showed almost no fluorescence signal. In contrast to [[Part:BBa_K1045013|BBa_K1045013]], [[Part:BBa_K1045017|BBa_K1045017]]
codes for
DarR. The low fluorescence signal suggests that DarR was synthesized and fully active as a repressor that prevents ''gfp'' transcription by binding to the DarR operator. Hence, DarR seems to act as a strong repressor in ''E. coli'' even in the absence of c-di-AMP.
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To characterize the DarR reporter system, the ''E. coli'' strain BL21 was transformed either with [[Part:BBa_K1045017|BBa_K1045017]] or with [[Part:BBa_K1045013|BBa_K1045013]] as a control. In [[Part:BBa_K1045013|BBa_K1045013]], ''gfp'' is placed downstream of a strong promoter and the DarR operator. This vector does not encode DarR. The strong fluorescence signal of cells transformed with [[Part:BBa_K1045013|BBa_K1045013]] indicated that GFP was produced. However, when transformed with [[Part:BBa_K1045017|BBa_K1045017]] ('''Fig. 1'''), the bacteria showed almost no fluorescence signal. In contrast to [[Part:BBa_K1045013|BBa_K1045013]], [[Part:BBa_K1045017|BBa_K1045017]]
encodes the repressor
DarR. The low fluorescence signal suggests that DarR was synthesized and fully active as a repressor that prevents ''gfp'' transcription by binding to the DarR operator. Hence, DarR seems to act as a strong repressor in ''E. coli'' even in the absence of c-di-AMP.
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[[File:-DarR.jpg|420px|thumb|'''Fig. 1.''': ''Top'': ''E. coli'' cells that harbor plasmid encoding [[Part:BBa_K1045013|BBa_K1045013]] show a strong green fluorescence signal when analyzed with a fluorescence microscope. ''Bottom'': ''E. coli'' harboring the plasmid that contains the complete DarR reporter system [[Part:BBa_K1045017|BBa_K1045017]] do not emit a green fluorescence signal. Both pictures represent merges of a bright field image and a GFP channel image. The exposure time used to record GFP fluorescence was in both cases 2 seconds. [[File:+DarR.jpg|420px]]|center]]
[[File:-DarR.jpg|420px|thumb|'''Fig. 1.''': ''Top'': ''E. coli'' cells that harbor plasmid encoding [[Part:BBa_K1045013|BBa_K1045013]] show a strong green fluorescence signal when analyzed with a fluorescence microscope. ''Bottom'': ''E. coli'' harboring the plasmid that contains the complete DarR reporter system [[Part:BBa_K1045017|BBa_K1045017]] do not emit a green fluorescence signal. Both pictures represent merges of a bright field image and a GFP channel image. The exposure time used to record GFP fluorescence was in both cases 2 seconds. [[File:+DarR.jpg|420px]]|center]]
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=====Plate Reader
-Data
=====
+
=====
Characterization of the Reporter System in a Multi-Well
Plate Reader=====
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We furthermore produced quantitative data characterizing
the growth
and the fluorescence over time
of the
BL21
''E. coli''
s we transformed with this construct.
+
In addition
the
microscopic analyses, we monitored
growth of the ''E. coli''
reporter strain and
the
green light that is emitted by
the
bacteria. The
plate reader experiments
were
performed to quantify the
capability
of
DarR of binding to
the DarR
operator ''
in
vivo
''.
The results of the experiments are summarized in
''
'Fig
.
2''' and '''Fig. 3'''. Growth was
measured at
a
wavelength
of
600 nm ('''Fig. 2''') and
light at a wavelength of
509 nm
was recorded to measure
GFP
production
('''Fig. 3'''). For each measurement, three technical and two biological replicates were
used
. The graphs show the mean value of the technical replicates and of
one
biological
replicate
. As
described
in the
figure
legend,
different amounts
of c-di-AMP
were added to the small-scall cultures in order
to test
whether
the
DNA-binding affinity of
DarR
is increased by
the
cyclic di-
nucleotide. Experimental setup: total
recording
time 21 h;
data were collected every
15 min;
the cells were grown at
37°C
in the
medium shaking
mode
;
cells were grown in LB medium supplemented in a sterile
96-well
micro
titer plate; Synergy Mx Monochromator-Based Multi-Mode Microplate Reader; Gen5 V2.01
Software (BioTek).
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The following graphs show
the
results of
the plate reader experiments performed to quantify the
strength
of the DarR
construct
in ''
E
.
coli
''.
Shown are growth curves
measured at
the
wavelength 600 nm
for the cell density
('''Fig. 2''') and 509 nm
for the
GFP ('''Fig. 3''')
, which is encoded in the construct
. For each measurement, three technical and two biological replicates were
set up
. The graphs show the mean value of the technical replicates and
one
of
the
biological
replicates
. As
written
in the legend,
a dilution series
of c-di-AMP
was set up
to test the DarR
reporter reaction to
the nucleotide. Experimental setup: total time 21 h; 15 min
measurement interval
; 37°C
,
medium shaking; 96-well titer plate; Synergy Mx Monochromator-Based Multi-Mode Microplate Reader; Gen5 V2.01
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[[File:DarR_Growth_cdiAMP.png|400px|thumb|'''Fig. 2''': ''Top'': Growth curve of
the
cells
with
the DarR construct; ''Bottom'': Growth curve of the GFP
Control
(
Cells transformed with
the reporter system
, but without
the repressor
DarR
).Please enlarge the pictures for better reading
. (click
on them
)
[[File:GFP_Control_Growth_cdiAMP.png|400px|]]|left]]
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[[File:DarR_Growth_cdiAMP.png|400px|thumb|'''Fig. 2''': ''Top'': Growth curve of cells
harboring
the DarR construct; ''Bottom'': Growth curve of the GFP
control
(
cells containing
the reporter system
that lack
the
DarR
repressor). Please enlarge the pictures for better reading
by clicking
on them
.
[[File:GFP_Control_Growth_cdiAMP.png|400px|]]|left]]
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[[File:DarR_Fluorescence_cdiAMP.png|400px|thumb|'''Fig. 2''': ''Top'': Fluorescence
curve
of the cells with the riboswitch construct; ''Bottom'': Fluorescence
curve
of the GFP
Control
. Please enlarge the pictures for better reading
(click
on them
)
.[[File:GFP_Control_Fluorescence_cdiAMP.png|400px|]]|right]]
+
[[File:DarR_Fluorescence_cdiAMP.png|400px|thumb|'''Fig. 2''': ''Top'': Fluorescence
signals
of the cells with the riboswitch construct; ''Bottom'': Fluorescence
signals
of the GFP
control
. Please enlarge the pictures for better reading
by clicking
on them.[[File:GFP_Control_Fluorescence_cdiAMP.png|400px|]]|right]]
<br style="clear:both;"/>
<br style="clear:both;"/>
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As
in
the
microscope experiments described
above, DarR prevented expression of the reporter, even without c-di-AMP. It
was observed
that the
presence
of c-di-AMP, regardless of the concentration used, had no effect on the ''gfp'' expression. This data indicated a high-affinity binding of DarR to its operator in ''E. coli'' in the abscence of c-di-AMP.
+
As
revealed by
the
microscopic studies (see
above
)
, DarR prevented expression of the reporter, even without c-di-AMP. It
has been reported previously
that
binding of DarR to its operator is stimulated by c-di-AMP. However, in our experiments
the
addition
of c-di-AMP, regardless of the concentration used, had no effect on the ''gfp'' expression. This data indicated a high-affinity binding of DarR to its operator in ''E. coli''
, even
in the abscence of c-di-AMP.
-
In conclusion, the experiments showed that the cells can grow with the construct, and that DarR is highly active as a repressor. In the future, mutagenesis of the operator sequence or the binding motive in the protein might
lower
the
strength of
the
repressor
. This
could make it possible
to
control DarR binding
to the
operator via
different c-di-AMP
concentrations
. Intermediate GFP expression leves for [[Part:BBa_K1045017|BBa_K1045017]] would allow
compound screening
to
find novel antibiotics directed against
c-di-AMP.
'''
In
contrast
, regarding the current binding
strenght
of DarR [[Part:BBa_K1045001|BBa_K1045001]] to
the
operator [[Part:BBa_K1045000|BBa_K1045000]],
these
two biobricks
could serve as
an "inverter".
Controlled
by an
inducable
promoter, DarR would
stop the
transcription of a gene connected to the DarR operator sequence
only upon induction
of
DarR expression
.'''
+
In conclusion, the experiments showed that the cells can grow with the construct, and that DarR is highly active as a repressor. In the future, mutagenesis of the operator sequence
(e.g., singly nucleotide exchanges)
or the binding motive in the protein might
decrease
the
interaction between DarR and
the
operator
. This
seems
to
be the appropriate approach
to
monitor DNA-binding activity of DarR in
the
presence of
different
amounts of
c-di-AMP. Intermediate GFP expression leves for [[Part:BBa_K1045017|BBa_K1045017]] would allow to
screen for compounds that compete with
c-di-AMP
for binding to non-essential and essential target proteins without killing Gram-negative bacteria that harbor the reporter system
.
Our system might be a promising starting point to identify novel antibiotics that kill Gram-positive pathogenic bacteria.
+
+
In
addition to this
, regarding the current binding
strength
of DarR
(
[[Part:BBa_K1045001|BBa_K1045001]]
)
to
its
operator
(
[[Part:BBa_K1045000|BBa_K1045000]]
)
,
the
two
novel
biobricks
might be used for the construction of
an "inverter".
In case the expression of ''darR'' were controlled
by an
inducible
promoter
(e.g., IPTG-dependent synthesis of DarR)
, DarR would
prevent
transcription of a gene
of interest (''goi'') that is
connected to the DarR operator sequence
in the presence
of
the inducer
.
By contrast, in the absence of the inducer, DarR is not formed and the
''
goi
'
' can be transcribed.
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