Difference between revisions of "Part:BBa K1045013:Experience"

Latest revision as of 13:43, 27 October 2013

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Applications of BBa_K1045013

This composite part is functional as indicated by the characterization experiments of our DarR reporter system BBa_K1045017. For this, we used BBa_K1045013 as a control plasmid. Without DarR, gfp was expressed. With DarR (but without c-di-AMP!), gfp expression was turned off. The characterization experiments for BBa_K1045017 are described below:

Microscope Data

For characterization, E. coli BL21 was transformed either with BBa_K1045017 or with BBa_K1045013 as a control. Both strains were grown in the abscence of c-di-AMP and subjected to fluorescence microscopy.

In BBa_K1045013, gfp is placed downstream of a strong promoter and the DarR operator. This vector does not encode for DarR. The strong fluorescence of the cells transformed with BBa_K1045013 (Fig. 1 top) indicated that GFP was expressed. However, when transformed with BBa_K1045017 (Fig. 1 bottom), the cells showed almost no fluorescence. In contrast to BBa_K1045013, BBa_K1045017 encodes for DarR. The low fluorescence suggested that DarR was expressed and active as a repressor down-regulating gfp transcription. Moreover, the results suggested that the control construct BBa_K1045013 was functional as it allowed GFP expression and regulation by DarR.

Fig. 1.: Top: E. coli transformed with a control plasmid encoding BBa_K1045013. Bottom: E. coli transformed with a plasmid harboring the DarR reporter system BBa_K1045017. Cells of both strains were cultured without c-di-AMP and analyzed by fluorescence microscopy. Both pictures represent merges of a bright field image and a GFP fluorescence image. The exposure time used to record GFP fluorescence was in both cases 2 seconds.

Experimental details: E. coli cells were grown in LB medium until log phase. A culture aliquot was prepared on slides covered with 1 % agarose (in water) and the cells observed under the fluorescence microscope. For all images, the same exposure time was used. Microscope: Axioskop 40 FL fluorescence microscope; Camera: digital camera AxioCam MRm; Software for image processing: AxioVision Rel version 4.8 (Carl Zeiss, Göttingen, Germany); Objective: Neofluar series objective (×100 primary magnification); Filter set: eGFP HC-Filterset (band-pass [BP] 472/30, FT 495, and long-pass [LP] 520/35; AHF Analysentechnik, Tübingen, Germany) for GFP detection.

Characterization of the Reporter System in a Multi-Well Plate Reader

We furthermore analyzed the growth and the fluorescence over time of the BL21 E. colis we transformed with the DarR reporter system construct BBa_K1045017. As a control, we employed E. coli cells harboring the BBa_K1045013 plasmid.

Using the plate reader, we quantified the strength of the DarR construct in E. coli. A dilution series of c-di-AMP (0, 50, 100, 150, 300, 500 and 1000 nmol c-di-AMP) was used to test the reaction of the DarR reporter system to the nucleotide.

Fig. 2 shows the growth curves recorded via the optical density (OD) at the wavelength 600 nm. The GFP fluorescence was measured at 509 nm over the time, as well. Since the fluorescence depends on the growth of the E. coli cells, the GFP fluorescence was normalized to the OD at 600 nm for each time point (Fig. 3). All graphs show the mean value of three technical replicates of one biological replicate. The error bars indicate the standard deviation.

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

Fig. 2: The growth of the E. coli cells was measured in a plate reader via the OD at 600 nm. To facilitate the differentiation between the growth phases, the OD at 600 nm is depicted in log scale. Top: E. coli cells carrying the control plasmid BBa_K1045013; Bottom: E. coli cells transformed with the DarR reporter system BBa_K1045017. The cells were cultured with c-di-AMP in different concentrations or without c-di-AMP. Please enlarge the pictures for better reading (click on them).

Fig. 3: The GFP fluorescence measured at 509 nm was normalized to the OD at 600 nm. Top: E. coli cells carrying the control plasmid BBa_K1045013; Bottom: E. coli cells transformed with the DarR reporter system BBa_K1045017. The cells were cultured with c-di-AMP in different concentrations or without c-di-AMP. Please enlarge the pictures for better reading (click on them).

As in the microscope experiments described above, the expression of the reporter was prevented (even without c-di-AMP), when DarR was encoded in the vector. This data suggested that DarR was expressed and active. In addition, the composite part BBa_K1045013 was functional as it allowed GFP expression that was shut down by DarR.

User Reviews

UNIQbd0d6a07fa8c765d-partinfo-00000000-QINU UNIQbd0d6a07fa8c765d-partinfo-00000001-QINU

@@ Line 6: / Line 6: @@
 ===Applications of BBa_K1045013===
-This composite part is functional as indicated by the characterization experiments of our DarR reporter system [[Part:BBa_K1045017|BBa_K1045017]]. For this, we used '''BBa_K1045013''' as a control plasmid. Without DarR, GFP was expressed. With DarR (but without c-di-AMP!), GFP expression was turned off. These characterization experiments are described below:
+This composite part is functional as indicated by the characterization experiments of our DarR reporter system [[Part:BBa_K1045017|BBa_K1045017]]. For this, we used '''BBa_K1045013''' as a control plasmid. Without DarR, ''gfp'' was expressed. With DarR (but without c-di-AMP!), ''gfp'' expression was turned off. The characterization experiments for [[Part:BBa_K1045017|BBa_K1045017]] are described below:
-== Microscope data ==
+== Microscope Data ==
-For characterization, ''E. coli'' BL21 was transformed either with [[Part:BBa_K1045017|BBa_K1045017]] or with [[Part:BBa_K1045013|BBa_K1045013]] as a control. Both strains were grown in the abscence of c-di-AMP and subjected to fluorescence microscopy.
+For characterization, ''E. coli'' BL21 was transformed either with [[Part:BBa_K1045017|BBa_K1045017]] or with '''BBa_K1045013''' as a control. Both strains were grown in the abscence of c-di-AMP and subjected to fluorescence microscopy.
-In [[Part:BBa_K1045013|BBa_K1045013]], ''gfp'' is placed downstream of a strong promoter and the DarR operator. This vector does not encode for DarR. The strong fluorescence of the cells transformed with [[Part:BBa_K1045013|BBa_K1045013]] ('''Fig. 1''' ''top'') indicated that GFP was expressed. However, when transformed with [[Part:BBa_K1045017|BBa_K1045017]] ('''Fig. 1''' ''bottom''), the cells showed almost no fluorescence. In contrast to [[Part:BBa_K1045013|BBa_K1045013]], [[Part:BBa_K1045017|BBa_K1045017]] encodes for DarR. The low fluorescence suggested that DarR was expressed and active as a repressor down-regulating ''gfp'' transcription. Moreover, the results suggested that the control construct '''BBa_K1045013''' was functional as it allowed GFP expression and regulation by DarR.
+In '''BBa_K1045013''', ''gfp'' is placed downstream of a strong promoter and the DarR operator. This vector does not encode for DarR. The strong fluorescence of the cells transformed with '''BBa_K1045013''' ('''Fig. 1''' ''top'') indicated that GFP was expressed. However, when transformed with [[Part:BBa_K1045017|BBa_K1045017]] ('''Fig. 1''' ''bottom''), the cells showed almost no fluorescence. In contrast to '''BBa_K1045013''', [[Part:BBa_K1045017|BBa_K1045017]] encodes for DarR. The low fluorescence suggested that DarR was expressed and active as a repressor down-regulating ''gfp'' transcription. Moreover, the results suggested that the control construct '''BBa_K1045013''' was functional as it allowed GFP expression and regulation by DarR.
-[[File:-DarR.jpg|420px|thumb|'''Fig. 1.''': ''Top'': ''E. coli'' transformed with a control plasmid encoding [[Part:BBa_K1045013|BBa_K1045013]]. ''Bottom'': ''E. coli'' transformed with a plasmid harboring the DarR reporter system [[Part:BBa_K1045017|BBa_K1045017]]. Cells of both strains were cultured without c-di-AMP and analyzed by fluorescence microscopy. Both pictures represent merges of a bright field image and a GFP fluorescence 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'' transformed with a control plasmid encoding '''BBa_K1045013'''. ''Bottom'': ''E. coli'' transformed with a plasmid harboring the DarR reporter system [[Part:BBa_K1045017|BBa_K1045017]]. Cells of both strains were cultured without c-di-AMP and analyzed by fluorescence microscopy. Both pictures represent merges of a bright field image and a GFP fluorescence image. The exposure time used to record GFP fluorescence was in both cases 2 seconds. [[File:+DarR.jpg|420px]]|center]]
-== Plate reader data ==
+'''Experimental details''':
+''E. coli'' cells were grown in LB medium until log phase. A culture aliquot was prepared on slides covered with 1 % agarose (in water) and the cells observed under the fluorescence microscope. For all images, the same exposure time was used. Microscope: Axioskop 40 FL fluorescence microscope; Camera: digital camera AxioCam MRm; Software for image processing: AxioVision Rel version 4.8 (Carl Zeiss, Göttingen, Germany); Objective: Neofluar series objective (×100 primary magnification); Filter set: eGFP HC-Filterset (band-pass [BP] 472/30, FT 495, and long-pass [LP] 520/35; AHF Analysentechnik, Tübingen, Germany) for GFP detection.
-We furthermore produced quantitative data characterizing the growth and the fluorescence over time of the BL21 ''E. coli''s we transformed with the DarR reporter system construct [[Part:BBa_K1045017|BBa_K1045017]]. As a control, we used ''E. coli'' cells harboring the [[Part:BBa_K1045013|BBa_K1045013]] plasmid.
+==Characterization of the Reporter System in a Multi-Well Plate Reader==
-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 done. 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 used to test the reaction of the DarR reporter system 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
-[[File:DarR_Growth_cdiAMP.png|400px|thumb|'''Fig. 2''': ''Top'': Growth curve of the ''E. coli'' cells carrying the DarR reporter system [[Part:BBa_K1045017|BBa_K1045017]]; ''Bottom'': Growth curve of ''E. coli'' cells transformed with the control plasmid [[Part:BBa_K1045013|BBa_K1045013]]. The cells were cultured with c-di-AMP in different concentrations or without c-di-AMP. The growth was measured via the OD at 600 nm in a plate reader. Please enlarge the pictures for better reading (click on them).[[File:GFP_Control_Growth_cdiAMP.png|400px|]]|left]]
+We furthermore analyzed the growth and the fluorescence over time of the BL21 ''E. coli''s we transformed with the DarR reporter system construct [[Part:BBa_K1045017|BBa_K1045017]]. As a control, we employed ''E. coli'' cells harboring the '''BBa_K1045013''' plasmid.
+Using the plate reader, we quantified the strength of the DarR construct in ''E. coli''. A dilution series of c-di-AMP (0, 50, 100, 150, 300, 500 and 1000 nmol c-di-AMP) was used to test the reaction of the DarR reporter system to the nucleotide.
+'''Fig. 2''' shows the growth curves recorded via the optical density (OD) at the wavelength 600 nm. The GFP fluorescence was measured at 509 nm over the time, as well. Since the fluorescence depends on the growth of the ''E. coli'' cells, the GFP fluorescence was normalized to the OD at 600 nm for each time point ('''Fig. 3'''). All graphs show the mean value of three technical replicates of one biological replicate. The error bars indicate the standard deviation.
+'''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
+[[File:DarR_1.png|400px|thumb|'''Fig. 2''': The growth of the ''E. coli'' cells was measured in a plate reader via the OD at 600 nm. To facilitate the differentiation between the growth phases, the OD at 600 nm is depicted in log scale. ''Top'': ''E. coli'' cells carrying the control plasmid '''BBa_K1045013'''; ''Bottom'': ''E. coli'' cells transformed with the DarR reporter system [[Part:BBa_K1045017|BBa_K1045017]]. The cells were cultured with c-di-AMP in different concentrations or without c-di-AMP. Please enlarge the pictures for better reading (click on them).[[File:DarR_2.png|400px|]]|center]]
+[[File:DarR_6.png|400px|thumb|'''Fig. 3''': The GFP fluorescence measured at 509 nm was normalized to the OD at 600 nm. ''Top'': ''E. coli'' cells carrying the control plasmid '''BBa_K1045013'''; ''Bottom'': ''E. coli'' cells transformed with the DarR reporter system [[Part:BBa_K1045017|BBa_K1045017]]. The cells were cultured with c-di-AMP in different concentrations or without c-di-AMP. Please enlarge the pictures for better reading (click on them).[[File:DarR_5.png|400px|]]|center]]
-[[File:DarR_Fluorescence_cdiAMP.png|400px|thumb|'''Fig. 2''': ''Top'': Fluorescence curve of the ''E. coli'' cells with the DarR reporter system [[Part:BBa_K1045017|BBa_K1045017]]; ''Bottom'': Fluorescence curve of ''E. coli'' cells transformed with the control plasmid [[Part:BBa_K1045013|BBa_K1045013]]. The cells were cultured with c-di-AMP in different concentrations or without c-di-AMP. The fluorescence was measured at 509 nm in a plate reader. Please enlarge the pictures for better reading (click on them).[[File:GFP_Control_Fluorescence_cdiAMP.png|400px|]]|right]]
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