Difference between revisions of "Part:BBa K1045017:Experience"
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===Applications of BBa_K1045017=== | ===Applications of BBa_K1045017=== | ||
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| − | + | ====The DarR Reporter System==== | |
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| + | =====Microscope Data===== | ||
| + | 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. | ||
| − | [[ | + | 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 might hint 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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| − | ''E. coli'' | + | |
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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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| − | + | =====Platereader 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 this construct. | |
| − | + | The following graphs show the results of the platereader 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 coded 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 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: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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| − | + | 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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| − | + | However, we showed that the cells can grow with the construct, as well as the tremendous strength 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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| − | + | '''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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===User Reviews=== | ===User Reviews=== | ||
Revision as of 16:16, 3 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
Microscope Data
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 BBa_K1045017 or 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 for DarR. The strong fluorescence of the cells transformed with BBa_K1045013 might indicate that GFP was expressed. However, when transformed with BBa_K1045017 (Fig. 1), the cells showed almost no fluorescence. In contrast to BBa_K1045013, BBa_K1045017 encodes for DarR. The low fluorescence might hint 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.
Platereader Data
We furthermore produced quantitative data characterizing the growth and the fluorescence over time of the BL21 E. colis we transformed with this construct. The following graphs show the results of the platereader 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 coded 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 21h; 15min measurement interval; 37°C, medium shaking; 96 plate well; Synergy Mx Monochromator-Based Multi-Mode Microplate Reader; Gen5 V2.01
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.
However, we showed that the cells can grow with the construct, as well as the tremendous strength 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.
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
UNIQ97a4cc3bb047cb6b-partinfo-00000000-QINU UNIQ97a4cc3bb047cb6b-partinfo-00000001-QINU