Part:BBa_K1045017:Experience

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Applications of BBa_K1045017

The DarR Reporter System

Microscope Data

As described on our [http://2013.igem.org/Team:Goettingen/Project Wiki], we designed a c-di-AMP sensing in vitro screening system in E. coli. With this, we can screen for future antibiotic substances targeting the signal molecule c-di-AMP. To characterize the DarR reporter system, E. coli was transformed either with BBa_K1045017 or BBa_K1045013 as a control.

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 might indicate that GFP was expressed. However, when transformed with BBa_K1045017 (Fig. 1), the cells showed almost no fluorescence. In contrast to BBa_K1045013, BBa_K1045017 encodes for DarR. The low fluorescence hinted 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.

Fig. 1.: Top: E. coli transformed with a plasmid encoding BBa_K1045013 shows a strong green fluorescence under the fluorescence microscope. Bottom: E. coli transformed with a plasmid harboring the DarR reporter system barely shows fluorescence.

Plate Reader-Data

We furthermore produced quantitative data characterizing the growth and the fluorescence over time of the BL21 E. colis we transformed with this construct. 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 set up. 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 set up to test the DarR reporter reaction to the nucleotide. Experimental setup: total time 21 h; 15 min measurement interval; 37°C, medium shaking; 96 plate well; 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, DarR shuts down expression of the reporter, even without c-di-AMP. It was observed that the presence of c-di-AMP, regardless of the concentration used, did not increase the binding affinity of DarR to the DarR operator. A setup with higher concentration of c-di-AMP was not performed due to time and financial shortcomings.

However, we showed that the cells can grow with the construct, as well as the high activity of DarR 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 would make it possible to get an intermediate result applicable to sense different c-di-AMP levels.

Furthermore, DarR with its current strenght, could serve as an "inverter". Connected to an inducable promoter, DarR would stop the transcription of a gene connected to its operator sequence only upon induction.

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