Part:BBa_K1045011:Experience

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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. The experiments are described in the following sections:

Microscope data

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, BL21E. 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. Both pictures represent merges of a bright field image and a gfp image. The exposure time used to record GFP fluorescence was in both cases 2 seconds.

Plate reader data

We furthermore produced quantitative data characterizing the growth and the fluorescence over time of the BL21 E. colis we transformed with the DarR reporter system construct BBa_K1045017. As a control, we used E. coli cells harboring the BBa_K1045013 plasmid. The following graphs show the results of the plate reader experiments performed to quantify the strength of the DarR construct in E. coli. Shown are growth curves measured at the wavelength 600 nm for the cell density (Fig. 2) and 509 nm for the GFP (Fig. 3), which is encoded in the construct. For each measurement, three technical and two biological replicates were done. 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 used to test the reaction of the DarR reporter system to the nucleotide. Experimental setup: total time 21 h; 15 min measurement interval; 37°C, medium shaking; 96-well titer plate; Synergy Mx Monochromator-Based Multi-Mode Microplate Reader; Gen5 V2.01

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)

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).

As in the microscope experiments described above, the expression of the reporter was prevented (even without c-di-AMP), when DarR was encoded in the vector. Hence, DarR seemed to be expressed via the regulatory part BBa_K1045011 indicating that the promoter is active.

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