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 vitro 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. This construct is shown in Fig.1 as an overlay picture (CFP and Throughlight). To prove the function of our in vitro sensor for c-di-AMP, we grew our cells on different conditions. Without and with the signal nucleotide and a third condition with polyamines, which were supposed to enhance the uptake of c-di-AMP (Fig. 1). Since we se no difference between the conditions, we assume the B. subtilis promoter as part of the riboswitch to be so strong, that the amount of c-di-AMP entering the cells is just not enough to shut down expression of the reporter.

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

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 10.000nmol was used to test how strong the affinity of the riboswitch is. Shown are a growth curve (Fig. 2), measured at OD600, and the emitted light strength of CFP (Fig. 3), which expression is under the control of the ydaO riboswitch. In addition to the c-di-AMP, polyamines were added (1 µl/ml, 1000x stock solution) 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 technical replicates of one of the biologicals. Experimental setup: total time 21h; 15min 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 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 do 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 shows 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 formation of the riboswitch. Due to time and financial issues, those theory was not tested.

However, we have shown, that the cells express the CFP reporter over exponential and stationary phase under a promoter from B. subtilis ydaO gene. We also showed, that E. coli is 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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