Our project is aimed at the development of a simple screening system, which allows the rapid identification and characterization of substances that disturb c-di-AMP homeostasis in pathogenic bacteria. By developing this system, we believe we can help scientist and pharmaceutical companies worldwide to find new and more effective antibiotics against Gram-positive pathogens, for which c-di-AMP is essential. The screening system will be established in the non- pathogenic bacterium E. coli.
Using given Biobricks and by creating new ones, we first wanted to construct a promoter-reporter fusion system which is under the control of DarR, an operator derived from Mycobacterium smegmatis. Only in the presents of c-di-AMP will this operator able to inhibit the transcription of the reporter gene GFP. The operator binding sequence will be placed between a constitutively active and the reporter gene. The activity of this reporter is rather to detect, since it emits green light if not inhibited. Via the addition of exogenous c-di-AMP, we will have to evaluate whether it is able to inhibit our promoter-reporter system.