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

Revision as of 17:50, 12 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 above, 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 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 BBa_K1045017 and that it is functional though additional upstream basepairs were added to this part.

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

UNIQ2bfd48aa970d8f5b-partinfo-00000000-QINU UNIQ2bfd48aa970d8f5b-partinfo-00000001-QINU

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 ===Applications of BBa_K1045011===
-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. In this system, ''gfp'' is placed under the control of the DarR operator [[Part:BBa_K1045000|BBa_K1045000]]. We saw that GFP expression was repressed when ''E. coli'' harbored a construct carrying DarR under the control of the promoter in [[Part:BBa_K1045011|BBa_K1045011]]. For more information, see main page of [[Part:BBa_K1045017|BBa_K1045017]] and our entry on the experience page of BBa_K1045017:
+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.
-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.
+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 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 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 BBa_K1045017 and that it is functional though additional upstream basepairs were added to this part.