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 consisting of the iGEM biobricks in E. coli (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: When there is c-di-AMP present, the c-di-AMP can act as ligand of the repressor gene DarR, activating tis function. DarR binds to its binding site, blocking the RNA polymerase, and therefore disrupt transcription. Then we are supposed to see no signal or reduced signal.

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