Part:BBa_K1045000:Experience
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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. 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.
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