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Applications of BBa_K1045017

The DarR Reporter System

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.

Microscope Data

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.

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 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. It seems also possible that the c-di-AMP amounts used might have been too low to see an even stronger repression of gfp expression, since the Kd of DarR was shown to be 2.3 µM (Zhang et al., 2013). In addition, E. coli might be unable to take up c-di-AMP, as the characterization experiments of BBa_K1045002 suggested.

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. single nucleotide exchanges) or the binding motive in the protein might decrease the interaction between DarR and the operator. This could make it possible to control DarR binding to the operator via different c-di-AMP concentrations. For this, a way allowing E. coli to take up c-di-AMP might have to be established first. This seems, however, time-consuming. A much faster approach might be to express a c-di-AMP-synthesizing diadenylate cyclase (DAC) in E. coli cells harboring the reporter system. Part BBa_K1045003 encodes for a truncated version of the DAC from Listeria monocytogenes, which is active in E. coli. Using this part, c-di-AMP could be generated in vivo.

Mutagenesis of the DarR operator or DarR seems to be an appropriate approach to achieve a differential regulation by DarR dependent on 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. In addition, when BBa_K1045017 is combined with a DAC, compounds could be identified which inhibit these essential enzymes. 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 is 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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