Difference between revisions of "Part:BBa K1045012:Experience"

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Applications of BBa_K1045012

This composite part seems to be funtional, as the characterization of our DarR reporter system BBa_K1045017 indicated. We saw that from the control plasmid BBa_K1045013, which harbors this part upstream of GFP coding sequence, GFP is expressed. This shows that the promoter is active in absence of DarR (BBa_K1045001). Moreover, expression of DarR shuts down gfp transcription indicating that the operator BBa_K1045000 is functional. The sections below describe the characterization experiments for the DarR reporter system BBa_K1045017.

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. In addition, these results showed that the promoter BBa_J23110 and the DarR operator BBa_K1045000 were functional in regulating GFP expression.

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.

Experimental details: E. coli cells were grown in LB medium until log phase. A culture aliquot was prepared on slides covered with 1 % agarose (in water) and the cells observed under the fluorescence microscope. For all images, the same exposure time was used. Microscope: Axioskop 40 FL fluorescence microscope; Camera: digital camera AxioCam MRm; Software for image processing: AxioVision Rel version 4.8 (Carl Zeiss, Göttingen, Germany); Objective: Neofluar series objective (×100 primary magnification); Filter set: eGFP HC-Filterset (band-pass [BP] 472/30, FT 495, and long-pass [LP] 520/35; AHF Analysentechnik, Tübingen, Germany) for GFP detection.

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 E. coli cells carrying the DarR reporter system BBa_K1045017; Bottom: Growth curve of E. coli cells transformed with the control plasmid BBa_K1045013. The cells were cultured with c-di-AMP in different concentrations or without c-di-AMP. The growth was measured via the OD at 600 nm in a plate reader. Please enlarge the pictures for better reading (click on them).

Fig. 2: Top: Fluorescence curve of the E. coli cells with the DarR reporter system BBa_K1045017; Bottom: Fluorescence curve of E. coli cells transformed with the control plasmid BBa_K1045013. The cells were cultured with c-di-AMP in different concentrations or without c-di-AMP. The fluorescence was measured at 509 nm in a plate reader. Please enlarge the pictures for better reading (click on them).

As in the microscope experiments described above, the expression of the reporter was only prevented, when DarR was encoded in the vector. Hence, the promoter BBa_J23110 and the DarR operator BBa_K1045000 used to control the GFP levels were functional.

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