Part:BBa_K1045002:Experience

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Applications of BBa_K1045002

The Riboswitch Reporter System

Microscope Data

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. With this, we can screen for future antibiotic substances targeting the signal molecule c-di-AMP.

In order to do so, we combined the ydaO riboswitch fom B. subtilis with a CFP reporter. E. coli cells transformed with this construct were characterized by fluorescence microscopy. We grew our cells under different conditions: without and with c-di-AMP and with c-di-AMP plus polyamines, which were supposed to allow the uptake of c-di-AMP (Fig. 1, Oppenheimer-Shaaman et al., 2011).

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: Filter set 47 (BP 436/20, FT 455, and LP 480/40; Carl Zeiss) for CFP detection.

Fig. 1. left: YdaO without c-di-AMP, center: Ydao + c-di-AMP, right: YdaO + c-di-AMP + polyamine. 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.

Since we saw no difference between the conditions, we assumed the B. subtilis promoter as part of the riboswitch to be so strong, that the amount of c-di-AMP entering the cells was just not enough to shut down expression of the reporter.

In order to achieve 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.

Platereader 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 represent the results of two plate reader experiments performed to quantify the strength of the ydaO riboswitch construct. In this setup, a dilution series of c-di-AMP ranging from 0 to 10.000 nmol was used to test how strong the affinity of the riboswitch is. Shown are a growth curve recorded via the OD at 600 nm (Fig. 2), and the emission strength of CFP (Fig. 3), which expression is under the control of the ydaO riboswitch. 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. For each sample two biological and two technical replicates were made. The graphs show the mean values of the technical replicates of one of the biologicals. 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: Top: Growth curve of the cells with the riboswitch construct; Bottom: Growth curve of the CFP Control (Cells transformed with CFP but without the riboswitch in front of it).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 CFP Control. 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 used concentrations of c-di-AMP had no measurable effect on the riboswitch either. The single riboswitch replicate, that showed lower fluorescence (highest concentration) could not be replicated. We assume this to be an artifact or a pipetting mistake. It is believed that even higher amounts of c-di-AMP are necessary to change the the riboswitch secondary structure such that cfp is not expressed. Due to time and financial issues, those hypotheses were not tested.

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

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