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

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(→‎Applications of BBa_K1045000)
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We used this Biobrick in our DarR reporter system ([[Part:BBa_K1045017|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 ([[Part:BBa_K1045001|BBa_K1045001]]) even in the absence of c-di-AMP.
 
We used this Biobrick in our DarR reporter system ([[Part:BBa_K1045017|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 ([[Part:BBa_K1045001|BBa_K1045001]]) even in the absence of c-di-AMP.
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== Microscope data ==
  
 
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.
 
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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[[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]]
 
[[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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== 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 DarR reporter system construct [[Part:BBa_K1045017|BBa_K1045017]].
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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_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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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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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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'''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.'''
  
 
===User Reviews===
 
===User Reviews===

Revision as of 13:34, 16 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

The experimental setup used for characterization involved two different E. coli strains: E. coli was transformed either with BBa_K1045017 or 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 plasmid encoding 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. +DarR.jpg


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 DarR reporter system construct BBa_K1045017. 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

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)GFP Control Growth cdiAMP.png
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).GFP Control Fluorescence cdiAMP.png


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.

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.

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.

User Reviews

UNIQ8b98b1fb436cb712-partinfo-00000000-QINU UNIQ8b98b1fb436cb712-partinfo-00000001-QINU