Part:BBa_K1045000:Experience
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Applications of BBa_K1045000
We used this Biobrick in our DarR reporter system (BBa_K1045017). When characterizing this system in E. coli, we noticed that the DarR operator sequence as it is in BBa_K1045000 seems to be strongly bound by DarR (BBa_K1045001) even in the absence of c-di-AMP.
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. Hence, DarR seems to act as a strong repressor in E. coli even in the absence of cyclic di-AMP.
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 (Ă100 primary magnification); Filter set: eGFP HC-Filterset (band-pass [BP] 472/30, FT 495, and long-pass [LP] 520/35; AHF Analysentechnik, TĂŒbingen, 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. 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.
Fig. 2 shows the growth curves recorded via the optical density (OD) at the wavelength 600 nm. Fig. 3 illustrates the GFP fluorescence measured at 509 nm. Since the fluorescence depends on the growth of the E. coli cells, the GFP fluorescence was normalized to the OD at 600 nm (Fig. 4).
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
As in the microscope experiments described above, DarR prevented expression of the reporter, even without c-di-AMP. It was observed that the presence of c-di-AMP, regardless of the concentration used, had no effect on the gfp expression. This data indicated a high-affinity binding of DarR to its operator in E. coli in the abscence of c-di-AMP.
In conclusion, the experiments showed that the cells can grow with the construct, and that DarR is highly active as a repressor. In the future, mutagenesis of the operator sequence or the binding motive in the protein might lower the strength of the repressor. This could make it possible to control DarR binding to the operator via c-di-AMP. Regarding the current binding strenght of DarR BBa_K1045001 to the operator BBa_K1045000, these two biobricks could serve as an "inverter". Controlled by an inducable promoter, DarR would stop the transcription of a gene connected to the DarR operator sequence only upon induction of DarR expression.
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