Part:BBa_K1045002:Experience

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Applications of BBa_K1045002

The Riboswitch Reporter System

As described on our [http://2013.igem.org/Team:Goettingen/Project Wiki], we designed a c-di-AMP-sensing in vivo screening system in E. coli. This system could be used to screen for future antibiotic substances targeting the signal molecule c-di-AMP. To construct the riboswitch reporter system, we combined the ydaO riboswitch fom B. subtilis with cfp as a reporter gene. The system was characterized as described below.

Microscope Data

E. coli cells transformed with BBa_K1045002 were characterized by fluorescence microscopy (Fig. 1). We grew the cells under different conditions: without and with 1 µg/ml c-di-AMP and with c-di-AMP (1 µg/ml) plus polyamines. Polyamines served to allow the uptake of c-di-AMP (Oppenheimer-Shaaman et al., 2011).

Experimental details: E. coli cells were grown in LB medium. 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: Filter set 47 (BP 436/20, FT 455, and LP 480/40; Carl Zeiss) for CFP detection.

Fig. 1. E. coli cells transformed with BBa_K1045002 were cultivated under different conditions and analyzed by fluorescence microscopy. Left: in absence of c-di-AMP, center: in presence of c-di-AMP (1 µg/ml), right: in presence of c-di-AMP (1 µg/ml) and polyamines. All pictures represent merges of a bright field image and a CFP fluorescence image. The exposure time used to record CFP fluorescence was in all cases 1 second.

Ideally, in the presence of c-di-AMP, cfp expression should be prevented by the riboswitch. However, we saw no difference between cells grown with or without c-di-AMP. Nevertheless, the strong fluorescence indicated that the ydaO promoter from B. subtilis driving the expression of the riboswitch reporter system is highly active. The high promoter activity might even explain why we saw no difference between the different growth conditions: The strong promoter could lead to high RNA levels. Compared to the high RNA levels, the c-di-AMP amounts entering the cells might have been too low. Thus, the expected premature transcripional termination mediated by c-di-AMP and the ydaO riboswitch could not have been visible. In order to achieve premature termination of transcription (e.g. in order to use this biobrick as a "negative inductor"), we suggest our shorter version of the riboswitch (BBa_K1045005, the riboswitch without its native promoter) combined with a weaker promoter. Alternatively, the c-di-AMP amounts could be increased. It could be possible, as well, that E. coli is unable to take up c-di-AMP. Hence, one should analyze E. coli regarding its ability to take up c-di-AMP. Another approach could 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.

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 riboswitch reporter system BBa_K1045002. As a control, we employed E. coli cells transformed with the plasmid carrying the cfp gene, but lacking the control elements for CFP expression (BBa_E0020).

The plate reader was used to quantify the strength of the ydaO riboswitch construct. In this setup, a dilution series of c-di-AMP ranging from 0 to 10000 nmol was used to test how strong the affinity of the riboswitch is. In addition to the c-di-AMP, polyamines (1 µl/ml, 1000x stock solution) were added to series of samples to test if the uptake of c-di-AMP into E. coli could be enhanced by this additive. The graphs show the mean values with the standard deviation of two technical replicates of one biological replicate.

Fig. 2 shows the growth curves recorded via the OD at 600 nm. The CFP fluorescence was measured at 480 nm and normalized to the cell density (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_E0020; Bottom: E. coli cells transformed with the riboswitch reporter system BBa_K1045002. The cells were cultured with c-di-AMP in different concentrations or without c-di-AMP. The dilution series was done with or without polyamines (PA, 1 µl/ml, 1000x stock solution). Note that for Fig. 2 top, some error bars are only shown as positive error bars. Since the corresponding negative error bars reached into the negative number range, it was not possible to depict them in a log scale diagram. Please enlarge the pictures for better reading (click on them).

Fig. 3: The CFP fluorescence measured at 480 nm was normalized to the OD at 600 nm. Top: E. coli cells carrying the control plasmid BBa_E0020; Bottom: E. coli cells transformed with the riboswitch reporter system BBa_K1045002. The cells were cultured with c-di-AMP in different concentrations or without c-di-AMP. The dilution series was done with or without polyamines (PA, 1 µl/ml, 1000x stock solution). Please enlarge the pictures for better reading (click on them).

It was observed that the polyamines did not influence the uptake of c-di-AMP into the cells in one way or the other. The concentrations of c-di-AMP tested had no measurable effect on the riboswitch either. The single riboswitch replicate, that showed lower fluorescence (highest concentration) might not be significant as the error bars indicate. We assume this to be an artifact or a pipetting mistake.

There are two possible explanations why we saw no effect of c-di-AMP on cfp expression: (1) the riboswitch vs. c-di-AMP levels are unbalanced. This could be caused by the high activity of the ydaO promoter leading to very high amounts of riboswitch compared to the c-di-AMP amounts used. Alternatively, the exogenously added c-di-AMP amounts were too low. (2) E. coli is unable to take up c-di-AMP even with polyamines. Thus, future experiments have to involve (1) a riboswitch reporter system expressed from a weaker promoter and/or higher c-di-AMP concentrations; and (2) analysis of c-di-AMP uptake by E. coli and/or expression of the c-di-AMP-synthesizing diadenylate cyclases (e.g. BBa_K1045003, a truncated version of the Listeria monocytogenes DAC) in E. coli cells harboring the reporter system.

In conlusion, we showed that the E. coli cells expressed the CFP reporter over stationary phase under a promoter from B. subtilis ydaO gene. We also showed, that E. coli was not harmed or hindered in its growth, allowing the reporter system to be used without the danger of killing our host.

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