Difference between revisions of "Part:BBa K1045002:Experience"

(Applications of BBa_K1045002)
(Plate reader data)
 
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====The Riboswitch Reporter System====
 
====The Riboswitch Reporter System====
  
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As described on our [http://2013.igem.org/Team:Goettingen/Project Wiki], we designed a c-di-AMP-sensing ''in vivo'' screening system in ''E. coli''. This system could be used to screen for future antibiotic substances targeting the signal molecule c-di-AMP. To construct the riboswitch reporter system, we combined the ''ydaO'' riboswitch fom ''B. subtilis'' with ''cfp'' as a reporter gene. The system was characterized as described below.
  
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==Microscope Data==
  
=====Microscope Data=====
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''E. coli'' cells transformed with '''BBa_K1045002''' were characterized by fluorescence microscopy ('''Fig. 1'''). We grew the cells under different conditions: without and with 1 µg/ml c-di-AMP and with c-di-AMP (1 µg/ml) plus polyamines. Polyamines served to allow the uptake of c-di-AMP (Oppenheimer-Shaaman ''et al''., 2011).
  
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.
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'''Experimental details''':
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''E. coli'' cells were grown in LB medium. 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: Filter set 47 (BP 436/20, FT 455, and LP 480/40; Carl Zeiss) for CFP detection.
  
In order to do so, we combined the ydaO riboswitch fom ''B. subtilis'' with a CFP reporter. This construct is shown in Fig.1 as an overlay picture (CFP and Throughlight). To prove the function of our ''in vitro'' sensor for c-di-AMP, we grew our cells on different conditions. Without and with the signal nucleotide and a third condition with polyamines, which were supposed to enhance the uptake of c-di-AMP.
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[[File:YdaO_complete2.jpg|1000px|center|thumb|'''Fig. 1.''' ''E. coli'' cells transformed with '''BBa_K1045002''' were cultivated under different conditions and analyzed by fluorescence microscopy. ''Left'': in absence of c-di-AMP, ''center'': in presence of c-di-AMP (1 µg/ml), ''right'': in presence of c-di-AMP (1 µg/ml) and polyamines. All pictures represent merges of a bright field image and a CFP fluorescence image. The exposure time used to record CFP fluorescence was in all cases 1 second.]]
Since we se no difference between the conditions, we assume the ''B. subtilis'' promoter as part of the riboswitch to be so strong, that the amount of c-di-AMP entering the cells is just not enough to shut down expression of the reporter.
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[[File:YdaO_complete2.jpg|1000px|center|thumb|'''Fig. 1.''' ''left'': YdaO without c-di-AMP, ''center'': Ydao + c-di-AMP, ''right'': YdaO + c-di-AMP + polyamine]]
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Ideally, in the presence of c-di-AMP, ''cfp'' expression should be prevented by the riboswitch. However, we saw no difference between cells grown with or without c-di-AMP. Nevertheless, the strong fluorescence indicated that the ''ydaO'' promoter from ''B. subtilis'' driving the expression of the riboswitch reporter system is highly active. The high promoter activity might even explain why we saw no difference between the different growth conditions: The strong promoter could lead to high RNA levels. Compared to the high  RNA levels, the c-di-AMP amounts entering the cells might have been too low. Thus, the expected premature transcripional termination mediated by c-di-AMP and the ''ydaO'' riboswitch could not have been visible. '''In order to achieve premature termination of transcription (e.g. in order to use this biobrick as a "negative inductor"), we suggest our shorter version of the riboswitch ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K1045005 BBa_K1045005, the riboswitch without its native promoter]) combined with a weaker promoter.''' Alternatively, the c-di-AMP amounts could be increased. It could be possible, as well, that'' E. coli'' is unable to take up c-di-AMP. Hence, one should analyze ''E. coli'' regarding its ability to take up c-di-AMP. Another approach could 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''.
  
'''In order to achieve termination of transcription (e.g. in order to use this biobrick as a "negative inductor"), we suggest our shorter version of the riboswitch ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K1045005 BBa_K1045005, the riboswitch without its native promoter]) combined with a weaker promoter.'''
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==Plate reader data==
  
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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 riboswitch reporter system '''BBa_K1045002'''. As a control, we employed ''E. coli'' cells transformed with the plasmid carrying the ''cfp'' gene, but lacking the control elements for CFP expression ([[Part:BBa_E0020|BBa_E0020]]).
  
=====Platereader Data=====
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The plate reader was used to quantify the strength of the ''ydaO'' riboswitch construct. In this setup, a dilution series of c-di-AMP ranging from 0 to 10000 nmol was used to test how strong the affinity of the riboswitch is. In addition to the c-di-AMP, polyamines (1 µl/ml, 1000x stock solution) were added to series of samples to test if the uptake of c-di-AMP into ''E. coli'' could be enhanced by this additive. The graphs show the mean values with the standard deviation of two technical replicates of one biological replicate.
  
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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'''Fig. 2''' shows the growth curves recorded via the OD at 600 nm. The CFP fluorescence was measured at 480 nm and normalized to the cell density ('''Fig. 3''').  
  
The following graphs represent the results of two plate reader experiments performed to quantify the strength of the ''ydaO'' riboswitch construct. In this setup, a dilution series of c-di-AMP ranging from 0 to 10.000nmol was used to test how strong the affinity of the riboswitch is. Shown are a growth curve (Fig. 2), measured at OD600, and the emitted light strength of CFP (Fig. 3), which expression is under the control of the ydaO riboswitch. In addition to the c-di-AMP, polyamines were added (1 µl/ml, 1000x stock solution) series of samples to test if the uptake of c-di-AMP into E. coli could be enhanced by this additive.
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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
For each sample two biological and two technical replicates were made. The graphs show the mean values of technical replicates of one of the biologicals. Experimental setup: total time 21h; 15min 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:Riboswitch_Growth.png|400px|thumb|'''Fig. 2''': ''Top'': Growth curve of the cells with the riboswitch construct; ''Bottom'': Growth curve of the CFP Control (Cells transformed with CFP but without the riboswitch in front of it).Please enlarge the pictures for better reading. (click on them)[[File:CFP_Control_Growth.png|400px|]]|left]]
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[[File:Riboswitch_4.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_E0020|BBa_E0020]]; ''Bottom'': ''E. coli'' cells transformed with the riboswitch reporter system '''BBa_K1045002'''. The cells were cultured with c-di-AMP in different concentrations or without c-di-AMP. The dilution series was done with or without polyamines (PA, 1 µl/ml, 1000x stock solution).
  
[[File:Riboswitch_Fluorescence.png|400px|thumb|'''Fig. 2''': ''Top'': Fluorescence curve of the cells with the riboswitch construct; ''Bottom'': Fluorescence curve of the CFP Control. Please enlarge the pictures for better reading (click on them).[[File:CFP_Control_Fluorescence.png|400px|]]|right]]
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Note that for Fig. 2 ''top'', some error bars are only shown as positive error bars. Since the corresponding negative error bars reached into the negative number range, it was not possible to depict them in a log scale diagram. Please enlarge the pictures for better reading (click on them).[[File:Riboswitch_3.png|400px|]]|center]]
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[[File:Riboswitch_2.png|400px|thumb|'''Fig. 3''': The CFP fluorescence measured at 480 nm was normalized to the OD at 600 nm. ''Top'': ''E. coli'' cells carrying the control plasmid [[Part:BBa_E0020|BBa_E0020]]; ''Bottom'': ''E. coli'' cells transformed with the riboswitch reporter system '''BBa_K1045002'''. The cells were cultured with c-di-AMP in different concentrations or without c-di-AMP. The dilution series was done with or without polyamines (PA, 1 µl/ml, 1000x stock solution). Please enlarge the pictures for better reading (click on them).[[File:Riboswitch_1.png|400px|]]|center]]
  
 
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<br>It was observed that the polyamines do not influence the uptake of c-di-AMP into the cells in one way or the other. The used concentrations of c-di-AMP had no measurable effect on the riboswitch either. The single riboswitch replicate, that shows lower fluorescence (highest concentration) could not be replicated. We assume this to be an artifact or a pipetting mistake. It is believed that even higher amounts of c-di-AMP are necessary to change the formation of the riboswitch. Due to time and financial issues, those theory was not tested.<br />
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<br>It was observed that the polyamines did not influence the uptake of c-di-AMP into the cells in one way or the other. The concentrations of c-di-AMP tested had no measurable effect on the riboswitch either. The single riboswitch replicate, that showed lower fluorescence (highest concentration) might not be significant as the error bars indicate. We assume this to be an artifact or a pipetting mistake.<br />
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There are two possible explanations why we saw no effect of c-di-AMP on ''cfp'' expression: (1) the riboswitch vs. c-di-AMP levels are unbalanced. This could be caused by the high activity of the ''ydaO'' promoter leading to very high amounts of riboswitch compared to the c-di-AMP amounts used. Alternatively, the exogenously added c-di-AMP amounts were too low. (2) ''E. coli'' is unable to take up c-di-AMP even with polyamines. Thus, future experiments have to involve (1) a riboswitch reporter system expressed from a weaker promoter and/or higher c-di-AMP concentrations; and (2) analysis of c-di-AMP uptake by ''E. coli'' and/or expression of the c-di-AMP-synthesizing diadenylate cyclases (e.g. [[Part:BBa_K1045003|BBa_K1045003]], a truncated version of the ''Listeria monocytogenes'' DAC) in ''E. coli'' cells harboring the reporter system.
  
However, we have shown, that the cells express the CFP reporter over exponential and stationary phase under a promoter from ''B. subtilis'' ydaO gene. We also showed, that ''E. coli'' is not harmed or hindered in its growth, even under high concentrations of c-di-AMP, allowing it to be used in our screening system without the danger of killing our host.
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In conlusion, we showed that the ''E. coli'' cells expressed the CFP reporter over stationary phase under a promoter from ''B. subtilis'' ''ydaO'' gene. We also showed, that ''E. coli'' was not harmed or hindered in its growth, allowing the reporter system to be used without the danger of killing our host.
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 19:48, 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_K1045002

The Riboswitch Reporter System

As described on our [http://2013.igem.org/Team:Goettingen/Project Wiki], we designed a c-di-AMP-sensing in vivo screening system in E. coli. This system could be used to screen for future antibiotic substances targeting the signal molecule c-di-AMP. To construct the riboswitch reporter system, we combined the ydaO riboswitch fom B. subtilis with cfp as a reporter gene. The system was characterized as described below.

Microscope Data

E. coli cells transformed with BBa_K1045002 were characterized by fluorescence microscopy (Fig. 1). We grew the cells under different conditions: without and with 1 µg/ml c-di-AMP and with c-di-AMP (1 µg/ml) plus polyamines. Polyamines served to allow the uptake of c-di-AMP (Oppenheimer-Shaaman et al., 2011).

Experimental details: E. coli cells were grown in LB medium. 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: Filter set 47 (BP 436/20, FT 455, and LP 480/40; Carl Zeiss) for CFP detection.

Fig. 1. E. coli cells transformed with BBa_K1045002 were cultivated under different conditions and analyzed by fluorescence microscopy. Left: in absence of c-di-AMP, center: in presence of c-di-AMP (1 µg/ml), right: in presence of c-di-AMP (1 µg/ml) and polyamines. All pictures represent merges of a bright field image and a CFP fluorescence image. The exposure time used to record CFP fluorescence was in all cases 1 second.

Ideally, in the presence of c-di-AMP, cfp expression should be prevented by the riboswitch. However, we saw no difference between cells grown with or without c-di-AMP. Nevertheless, the strong fluorescence indicated that the ydaO promoter from B. subtilis driving the expression of the riboswitch reporter system is highly active. The high promoter activity might even explain why we saw no difference between the different growth conditions: The strong promoter could lead to high RNA levels. Compared to the high RNA levels, the c-di-AMP amounts entering the cells might have been too low. Thus, the expected premature transcripional termination mediated by c-di-AMP and the ydaO riboswitch could not have been visible. In order to achieve premature termination of transcription (e.g. in order to use this biobrick as a "negative inductor"), we suggest our shorter version of the riboswitch (BBa_K1045005, the riboswitch without its native promoter) combined with a weaker promoter. Alternatively, the c-di-AMP amounts could be increased. It could be possible, as well, that E. coli is unable to take up c-di-AMP. Hence, one should analyze E. coli regarding its ability to take up c-di-AMP. Another approach could 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.

Plate reader data

We furthermore produced quantitative data characterizing the growth and the fluorescence over time of the BL21 E. colis we transformed with the riboswitch reporter system BBa_K1045002. As a control, we employed E. coli cells transformed with the plasmid carrying the cfp gene, but lacking the control elements for CFP expression (BBa_E0020).

The plate reader was used to quantify the strength of the ydaO riboswitch construct. In this setup, a dilution series of c-di-AMP ranging from 0 to 10000 nmol was used to test how strong the affinity of the riboswitch is. In addition to the c-di-AMP, polyamines (1 µl/ml, 1000x stock solution) were added to series of samples to test if the uptake of c-di-AMP into E. coli could be enhanced by this additive. The graphs show the mean values with the standard deviation of two technical replicates of one biological replicate.

Fig. 2 shows the growth curves recorded via the OD at 600 nm. The CFP fluorescence was measured at 480 nm and normalized to the cell density (Fig. 3).

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_E0020; Bottom: E. coli cells transformed with the riboswitch reporter system BBa_K1045002. The cells were cultured with c-di-AMP in different concentrations or without c-di-AMP. The dilution series was done with or without polyamines (PA, 1 µl/ml, 1000x stock solution). Note that for Fig. 2 top, some error bars are only shown as positive error bars. Since the corresponding negative error bars reached into the negative number range, it was not possible to depict them in a log scale diagram. Please enlarge the pictures for better reading (click on them).Riboswitch 3.png
Fig. 3: The CFP fluorescence measured at 480 nm was normalized to the OD at 600 nm. Top: E. coli cells carrying the control plasmid BBa_E0020; Bottom: E. coli cells transformed with the riboswitch reporter system BBa_K1045002. The cells were cultured with c-di-AMP in different concentrations or without c-di-AMP. The dilution series was done with or without polyamines (PA, 1 µl/ml, 1000x stock solution). Please enlarge the pictures for better reading (click on them).Riboswitch 1.png




It was observed that the polyamines did not influence the uptake of c-di-AMP into the cells in one way or the other. The concentrations of c-di-AMP tested had no measurable effect on the riboswitch either. The single riboswitch replicate, that showed lower fluorescence (highest concentration) might not be significant as the error bars indicate. We assume this to be an artifact or a pipetting mistake.

There are two possible explanations why we saw no effect of c-di-AMP on cfp expression: (1) the riboswitch vs. c-di-AMP levels are unbalanced. This could be caused by the high activity of the ydaO promoter leading to very high amounts of riboswitch compared to the c-di-AMP amounts used. Alternatively, the exogenously added c-di-AMP amounts were too low. (2) E. coli is unable to take up c-di-AMP even with polyamines. Thus, future experiments have to involve (1) a riboswitch reporter system expressed from a weaker promoter and/or higher c-di-AMP concentrations; and (2) analysis of c-di-AMP uptake by E. coli and/or expression of the c-di-AMP-synthesizing diadenylate cyclases (e.g. BBa_K1045003, a truncated version of the Listeria monocytogenes DAC) in E. coli cells harboring the reporter system.

In conlusion, we showed that the E. coli cells expressed the CFP reporter over stationary phase under a promoter from B. subtilis ydaO gene. We also showed, that E. coli was not harmed or hindered in its growth, allowing the reporter system to be used without the danger of killing our host.

User Reviews

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