Difference between revisions of "Part:BBa K1045011:Experience"

Revision as of 19:20, 15 October 2013

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Applications of BBa_K1045011

We used this part in our DarR reporter system BBa_K1045017. BBa_K1045011 was functional as our characterization experiments of BBa_K1045017 suggested.

BBa_K1045017 consists of two expression units. One expression unit serves to express the transcriptional repressor DarR, the other one drives expression of gfp. The gfp expression unit harbors a strong promoter and the DarR binding sequence. Hence, when DarR binds this sequence, gfp expression is supposed to be prevented. For the expression of DarR, the promoter BBa_K1045011 was used. In the experiment shown below, E. coli cells either transformed with the DarR reporter system or with BBa_K1045013 as a control vector (gfp expression unit only) were grown in the absence of c-di-AMP. Fluorescence microscopy revealed that the cells of the control strain were bright green indicating that GFP is expressed. When DarR was present in the vector, however, the E. coli cells were barely fluorescing. This suggests, that DarR is expresed from the promoter BBa_K1045011 in BBa_K1045017 and that it is functional though additional upstream basepairs were added to this part.

In conclusion, the part BBa_K1045011 is proven to be active. The fact that DarR is shutting down the gfp expression even in the absence of c-di-AMP (For further information and discussions, please visit here), implies a very high binding activity of DarR to the operator sequence.

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.

User Reviews

UNIQ73e54b0a22b702ac-partinfo-00000000-QINU UNIQ73e54b0a22b702ac-partinfo-00000001-QINU

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 We used this part in our DarR reporter system [[Part:BBa_K1045017|BBa_K1045017]]. [[Part:BBa_K1045011|BBa_K1045011]] was functional as our characterization experiments of [[Part:BBa_K1045017|BBa_K1045017]] suggested.
-[[Part:BBa_K1045017|BBa_K1045017]] consists of two expression units. One expression unit serves to express the transcriptional repressor DarR, the other one drives expression of ''gfp''. The gfp expression unit harbors a strong promoter and the DarR binding sequence. Hence, when DarR binds this sequence, ''gfp'' expression is supposed to be prevented. For the expression of DarR, the promoter '''BBa_K1045011''' was used. In the experiment shown above, ''E. coli'' cells either transformed with the  [[Part:BBa_K1045017|DarR reporter system]] or with [[Part:BBa_K1045013|BBa_K1045013]] as a control vector (''gfp'' expression unit only) were grown in the absence of c-di-AMP. Fluorescence microscopy revealed that the cells of the control cells were  bright green indicating that GFP is expressed. When DarR was present in the vector, however, the ''E. coli'' cells were barely fluorescing. This suggests, that DarR is expresed from the promoter '''BBa_K1045011''' in [[Part:BBa_K1045017|BBa_K1045017]] and that it is functional though additional upstream basepairs were added to this part.
+[[Part:BBa_K1045017|BBa_K1045017]] consists of two expression units. One expression unit serves to express the transcriptional repressor DarR, the other one drives expression of ''gfp''. The ''gfp'' expression unit harbors a strong promoter and the DarR binding sequence. Hence, when DarR binds this sequence, ''gfp'' expression is supposed to be prevented. For the expression of DarR, the promoter '''BBa_K1045011''' was used. In the experiment shown below, ''E. coli'' cells either transformed with the  [[Part:BBa_K1045017|DarR reporter system]] or with [[Part:BBa_K1045013|BBa_K1045013]] as a control vector (''gfp'' expression unit only) were grown in the absence of c-di-AMP. Fluorescence microscopy revealed that the cells of the control strain were  bright green indicating that GFP is expressed. When DarR was present in the vector, however, the ''E. coli'' cells were barely fluorescing. This suggests, that DarR is expresed from the promoter '''BBa_K1045011''' in [[Part:BBa_K1045017|BBa_K1045017]] and that it is functional though additional upstream basepairs were added to this part.
-'''In conclusion, the part '''BBa_K1045011''' is proven to be active. The fact that DarR is shutting down the ''gfp'' expression  even in the absence of c-di-AMP (For further information and discussions, please visit [[Part:BBa_K1045017|here]]), implies a very high activity.'''
+'''In conclusion, the part '''BBa_K1045011''' is proven to be active. The fact that DarR is shutting down the ''gfp'' expression  even in the absence of c-di-AMP (For further information and discussions, please visit [[Part:BBa_K1045017|here]]), implies a very high binding activity of DarR to the operator sequence.'''
 [[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]]