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, BL21 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 fluoresceing. This suggests, that DarR is expressed 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 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.

Characterization of the Reporter System in a Multi-Well Plate Reader

We furthermore analyzed 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 employed E. coli cells harboring the BBa_K1045013 plasmid. This plasmid carries only the GFP expression unit with a strong promoter and the DarR operator. It does not encode for DarR.

Using the plate reader, we quantified the strength of the DarR construct in E. coli. A dilution series of c-di-AMP (0, 50, 100, 150, 300, 500 and 1000 nmol c-di-AMP) was used to test the reaction of the DarR reporter system to the nucleotide.

Fig. 2 shows the growth curves recorded via the optical density (OD) at the wavelength 600 nm. The GFP fluorescence was measured at 509 nm over the time, as well. Since the fluorescence depends on the growth of the E. coli cells, the GFP fluorescence was normalized to the OD at 600 nm for each time point (Fig. 3). All graphs show the mean value of three technical replicates of one biological replicate. The error bars indicate the standard deviation.

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

Fig. 3: The GFP fluorescence measured at 509 nm was normalized to the OD at 600 nm. Top: E. coli cells carrying the control plasmid BBa_K1045013; Bottom: E. coli cells transformed with the DarR reporter system BBa_K1045017. The cells were cultured with c-di-AMP in different concentrations or without c-di-AMP. 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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