Part:BBa_K1045016:Experience

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Applications of BBa_K1045016

We used this part as a construction intermediate to obtain our DarR reporter system BBa_K1045017. Since this composite part was functional, we assume the construction intermediate BBa_K1045016 to be functional, as well. The characterization experiments for BBa_K1045017 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. In conclusion, BBa_K1045017 was active, suggesting that BBa_K1045016, too, encodes for a functional darR gene.

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 used 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.

Plate reader experiments were performed to quantify the strength of the DarR construct in E. coli. In these experiments, 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. Two biological replicates were done. For each biological replicate, three technical replicates were analyzed. The graphs below show the mean value of the technical replicates of one biological replicate. The error bars indicate the standard deviation.

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).

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. Though the DarR reporter system BBa_K1045017 could not be regulated by c-di-AMP, the expression units seem to be operative leading to DarR and GFP expression. This suggests, as well, that part BBa_K1045016 harbors a functional darR gene.

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