Difference between revisions of "Part:BBa K1045000:Experience"

(→‎Applications of BBa_K1045000)
(→‎Characterization of the Reporter System in a Multi-Well Plate Reader)
 
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== Microscope data ==
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== Microscope Data ==
  
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The experimental setup used for characterization involved two different ''E. coli'' strains: ''E. coli'' was transformed either with [[Part:BBa_K1045017|BBa_K1045017]] or [[Part:BBa_K1045013|BBa_K1045013]] as a control. Both strains were grown in the abscence of c-di-AMP and subjected to fluorescence microscopy.
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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.
  
 
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. Hence, DarR seems to act as a strong repressor in ''E. coli'' even in the absence of cyclic di-AMP.
 
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. Hence, DarR seems to act as a strong repressor in ''E. coli'' even in the absence of cyclic di-AMP.
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[[File:-DarR.jpg|420px|thumb|'''Fig. 1.''': ''Top'': ''E. coli'' transformed with a plasmid encoding [[Part:BBa_K1045013|BBa_K1045013]] shows a strong green fluorescence under the fluorescence microscope. ''Bottom'': ''E. coli'' transformed with a plasmid harboring the DarR reporter system barely shows fluorescence. [[File:+DarR.jpg|420px]]|center]]
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[[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]]
  
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'''Experimental details''':
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''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.
  
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==Characterization of the Reporter System in a Multi-Well Plate Reader==
  
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== Plate reader data ==
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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 [[Part:BBa_K1045013|BBa_K1045013]] plasmid. This plasmid carries only the GFP expression unit with a strong promoter and the DarR operator. It does not encode for DarR.
  
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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 the DarR reporter system construct [[Part:BBa_K1045017|BBa_K1045017]].
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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.
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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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'''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.
  
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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]]
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'''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_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 [[Part:BBa_K1045013|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]]
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[[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 [[Part:BBa_K1045013|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]]
  
 
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As in the Microscope experiments, DarR shuts down expression of the reporter, even without c-di-AMP. It was observed that the presence of c-di-AMP, regardless of the concentration used, did not increase the binding affinity of DarR to the DarR operator. A setup with higher concentration of c-di-AMP was not performed due to time and financial shortcomings.
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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. It seems also possible that the c-di-AMP amounts used might have been too low to see an even stronger repression of ''gfp'' expression, since the Kd of DarR was shown to be 2.3 µM (Zhang ''et al''., 2013). In addition, ''E. coli'' might be unable to take up c-di-AMP, as the characterization experiments of [[Part:BBa_K1045002|BBa_K1045002]] suggested.
  
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However, we 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 get an intermediate GFP expression level applicable to sense different c-di-AMP levels.
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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. For this, a way allowing ''E. coli'' to take up c-di-AMP might have to be established first. Alternatively, a c-di-AMP-synthesizing diadenylate cyclase (DAC) could be expressed in ''E. coli'' cells harboring the reporter system. Part [[Part:BBa_K1045003|BBa_K1045003]] encodes for a truncated version of the DAC from ''Listeria monocytogenes'', which is active in ''E. coli''. Using this part, c-di-AMP could be generated ''in vivo''.
  
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'''Furthermore, DarR with its current strenght, could serve as an "inverter". Connected to an inducable promoter, DarR would stop the transcription of a gene connected to its operator sequence only upon induction.'''
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'''Regarding the current binding strenght of DarR [[Part:BBa_K1045001|BBa_K1045001]] to the operator '''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.'''
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 19:09, 28 October 2013


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K1045000

We used this Biobrick in our DarR reporter system (BBa_K1045017). When characterizing this system in E. coli, we noticed that the DarR operator sequence as it is in BBa_K1045000 seems to be strongly bound by DarR (BBa_K1045001) even in the absence of c-di-AMP.


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. Hence, DarR seems to act as a strong repressor in E. coli even in the absence of cyclic di-AMP.


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. +DarR.jpg

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. This plasmid carries only the GFP expression unit with a strong promoter and the DarR operator. It does not encode for DarR.

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).DarR 2.png
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).DarR 5.png


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. It seems also possible that the c-di-AMP amounts used might have been too low to see an even stronger repression of gfp expression, since the Kd of DarR was shown to be 2.3 µM (Zhang et al., 2013). In addition, E. coli might be unable to take up c-di-AMP, as the characterization experiments of BBa_K1045002 suggested.

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. For this, a way allowing E. coli to take up c-di-AMP might have to be established first. Alternatively, a c-di-AMP-synthesizing diadenylate cyclase (DAC) could be expressed in E. coli cells harboring the reporter system. Part BBa_K1045003 encodes for a truncated version of the DAC from Listeria monocytogenes, which is active in E. coli. Using this part, c-di-AMP could be generated in vivo.

Regarding the current binding strenght of DarR BBa_K1045001 to the operator 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.

User Reviews

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