Team:Goettingen/Team/Reporter

Reporter Team

The activity of transcriptional regulators can be easily detected by monitoring the activity of a promoter that is fused to a reporter gene. Recently, in Mycobacterium smegmatis, a transcriptional repressor (DarR) was identified that is able to bind to a specific DNA sequence upon association with c-di-AMP. This led us to the idea of developing a screening system for potential drugs interfering with c-di-AMP biofunction. While many Gram-positive bacteria require c-di-AMP for their growth, this molecule is not synthesized by the Gram-negative bacterium E. coli. Thus, we intend to build a reporter system that is composed of existing and of novel biobricks that will be developed during the project (Figure 1).

Figure 1a: In the absence of c-di-AMP, DarR cannot bind to its binding site (operator) that is located downstream of the promoter. As a consequence the reporter gene is expressed and the cells synthesizing the green fluorescent protein (GFP) become fluorescent.

Figure 1b: In the presence of c-di-AMP, the signaling molecule stimulates DNA-binding activity of DarR and the c-di-AMP-DarR complex prevents transcription initiation by RNA polymerase. The E. coli cells do not produce GFP and are therefore non-fluorescent.

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