Difference between revisions of "Part:BBa K1045017:Experience"

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====The DarR Reporter System====
 
====The DarR Reporter System====
  
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=====Microscope Data=====
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As described on our [http://2013.igem.org/Team:Goettingen/Project Wiki], we designed a c-di-AMP-sensing screening system for the Gram-negative bacterium ''Escherichia coli''. Using this system, we can screen for substances that compete with c-di-AMP for binding to essential target proteins. Promising substances that bind these proteins might serve as a starting point for developing novel antibiotics that kill multi-resistant pathogenic bacteria. Since c-di-AMP is not present in ''E. coli'' and thus not needed for growth, we can sort out those substances that simply kill the bacteria and do not bind to c-di-AMP-binding proteins.
  
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As described on our [http://2013.igem.org/Team:Goettingen/Project Wiki], we designed a c-di-AMP sensing ''in vitro'' screening system in ''E. coli''. With this, we can screen for future antibiotic substances targeting the signal molecule c-di-AMP. To characterize the DarR reporter system, ''E. coli'' was transformed either with [[Part:BBa_K1045017|BBa_K1045017]] or [[Part:BBa_K1045013|BBa_K1045013]] as a control.
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==Microscope Data==
  
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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]] might indicate that GFP was expressed. However, when transformed with [[Part:BBa_K1045017|BBa_K1045017]] ('''Fig. 1'''), 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 hinted 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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To characterize the DarR reporter system, the ''E. coli'' strain BL21 was transformed either with '''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 '''BBa_K1045017''' ('''Fig. 1'''), the bacteria showed almost no fluorescence signal. In contrast to [[Part:BBa_K1045013|BBa_K1045013]], '''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'' 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'' 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 '''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=====
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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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We furthermore produced quantitative data characterizing the growth and the fluorescence over time of the BL21 ''E. coli''s we transformed with this construct.
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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 '''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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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 600nm for the cell density ('''Fig. 2''') and 509nm for the GFP ('''Fig. 3'''), which is encoded in the construct. For each measurement, 3 technical and 2 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 plate well; 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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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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'''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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'''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 '''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 '''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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[[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]]
 
  
 
<br style="clear:both;"/>
 
<br style="clear:both;"/>
  
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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 presents 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 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. 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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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. single nucleotide exchanges) or the binding motive in the protein might decrease the interaction between DarR and the operator. 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. This seems, however, time-consuming. A much faster approach might be to express a c-di-AMP-synthesizing diadenylate cyclase (DAC) 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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However, we showed that the cells can grow with the construct, as well as the high activity of DarR 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 would make it possible to get an intermediate result applicable to sense different c-di-AMP levels.
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Mutagenesis of the DarR operator or DarR seems to be an appropriate approach to achieve a differential regulation by DarR dependent on c-di-AMP. Intermediate GFP expression leves for '''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. In addition, when '''BBa_K1045017''' is combined with a DAC, compounds could be identified which inhibit these essential enzymes. Our system might be a promising starting point to identify novel antibiotics that kill Gram-positive pathogenic bacteria.  
  
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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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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'' is 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.
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 20:09, 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_K1045017

The DarR Reporter System

As described on our [http://2013.igem.org/Team:Goettingen/Project Wiki], we designed a c-di-AMP-sensing screening system for the Gram-negative bacterium Escherichia coli. Using this system, we can screen for substances that compete with c-di-AMP for binding to essential target proteins. Promising substances that bind these proteins might serve as a starting point for developing novel antibiotics that kill multi-resistant pathogenic bacteria. Since c-di-AMP is not present in E. coli and thus not needed for growth, we can sort out those substances that simply kill the bacteria and do not bind to c-di-AMP-binding proteins.

Microscope Data

To characterize the DarR reporter system, the E. coli strain BL21 was transformed either with BBa_K1045017 or with BBa_K1045013 as a control. In 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 BBa_K1045013 indicated that GFP was produced. However, when transformed with BBa_K1045017 (Fig. 1), the bacteria showed almost no fluorescence signal. In contrast to BBa_K1045013, 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.


Fig. 1.: Top: E. coli cells that harbor plasmid encoding 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 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. +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 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. 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 (e.g. single nucleotide exchanges) or the binding motive in the protein might decrease the interaction between DarR and the operator. 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. This seems, however, time-consuming. A much faster approach might be to express a c-di-AMP-synthesizing diadenylate cyclase (DAC) 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.

Mutagenesis of the DarR operator or DarR seems to be an appropriate approach to achieve a differential regulation by DarR dependent on c-di-AMP. Intermediate GFP expression leves for 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. In addition, when BBa_K1045017 is combined with a DAC, compounds could be identified which inhibit these essential enzymes. 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 (BBa_K1045001) to its operator (BBa_K1045000), the two novel biobricks might be used for the construction of an "inverter". In case the expression of darR is 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.

User Reviews

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