Part:BBa_K1045013:Experience

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Applications of BBa_K1045013

This composite part is functional as indicated by the characterization experiments of our DarR reporter system BBa_K1045017. For this, we used BBa_K1045013 as a control plasmid. Without DarR, GFP was expressed. With DarR (but without c-di-AMP!), GFP expression was turned off. These characterization experiments are described below:

Microscope data

For characterization, E. coli BL21 was transformed either with BBa_K1045017 or with 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. Moreover, the results suggested that the control construct BBa_K1045013 was functional as it allowed GFP expression and regulation by DarR.

Fig. 1.: Top: E. coli transformed with a control plasmid encoding BBa_K1045013. Bottom: E. coli transformed with a plasmid harboring the DarR reporter system BBa_K1045017. Cells of both strains were cultured without c-di-AMP and analyzed by fluorescence microscopy. Both pictures represent merges of a bright field image and a GFP fluorescence 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. This data suggested that DarR was expressed and active. In addition, the composite part BBa_K1045013 was functional as it allowed GFP expression that was shut down by DarR.

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