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

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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.
 
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.
  
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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]] indicated 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 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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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.
  
  

Revision as of 13:22, 16 October 2013


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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.

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.

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

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