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 screening system for the Gram-negative bacterium Escherichia coli. Using this system, we can screen for substances that compete with c-di-AMP for binding to essential target proteins. Promising substances that bind these proteins might serve as a starting point for developing novel antibiotics that kill multi-resistant pathogenic bacteria. Since c-di-AMP is not present in E. coli and thus not needed for growth, we can sort out those substances that simply kill the bacteria and do not bind to c-di-AMP-binding proteins!

To characterize the DarR reporter system, the E. coli strain BL21 was transformed either with BBa_K1045017 or with 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 DarR. The strong fluorescence signal of cells transformed with BBa_K1045013 indicated that GFP was produced. However, when transformed with BBa_K1045017 (Fig. 1), the bacteria showed almost no fluorescence signal. In contrast to BBa_K1045013, BBa_K1045017 encodes the repressor DarR. The low fluorescence signal suggests that DarR was synthesized and fully active as a repressor that prevents gfp transcription by binding to the DarR operator. Hence, DarR seems to act as a strong repressor in E. coli even in the absence of c-di-AMP.

Fig. 1.: Top: E. coli cells that harbor plasmid encoding BBa_K1045013 show a strong green fluorescence signal when analyzed with a fluorescence microscope. Bottom: E. coli harboring the plasmid that contains the complete DarR reporter system BBa_K1045017 do not emit a green fluorescence signal. Both pictures represent merges of a bright field image and a GFP channel image. The exposure time used to record GFP fluorescence was in both cases 2 seconds.

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

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. 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 revealed by the microscopic studies (see above), DarR prevented expression of the reporter, even without c-di-AMP. It has been reported previously that binding of DarR to its operator is stimulated by c-di-AMP. However, in our experiments the addition 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, even 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 (e.g., singly nucleotide exchanges) or the binding motive in the protein might decrease the interaction between DarR and the operator. This seems to be the appropriate approach to monitor DNA-binding activity of DarR in the presence of different amounts of c-di-AMP. Intermediate GFP expression leves for BBa_K1045017 would allow to screen for compounds that compete with c-di-AMP for binding to non-essential and essential target proteins without killing Gram-negative bacteria that harbor the reporter system. Our system might be a promising starting point to identify novel antibiotics that kill Gram-positive pathogenic bacteria.

In addition to this, regarding the current binding strength of DarR (BBa_K1045001) to its operator (BBa_K1045000), the two novel biobricks might be used for the construction of an "inverter". In case the expression of darR were controlled by an inducible promoter (e.g., IPTG-dependent synthesis of DarR), DarR would prevent transcription of a gene of interest (goi) that is connected to the DarR operator sequence in the presence of the inducer. By contrast, in the absence of the inducer, DarR is not formed and the goi can be transcribed.

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