Team:Goettingen/Team/Reporter

Reporter Team

The in vivo activity of a transcriptional regulator can be easily detected by monitoring the activity of a promoter that is fused to a reporter gene such as the green fluorescent protein (GFP). Recently, in Mycobacterium smegmatis, the transcriptional repressor DarR was identified (Zhang et al. 2013). DarR is capable of binding to a specific DNA sequence upon association with c-di-AMP. This led us to the idea of developing a powerful screening system to facilitate the identification of potential drugs interfering with c-di-AMP biofunction. While many Gram-positive bacteria (also pathogenic bacteria that cause severe human diseases) require c-di-AMP for growth, c-di-AMP is not synthesized by the Gram-negative bacterium Escherichia coli. Therefore, 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). As E. coli does not require c-di-AMP for growth the screening system can be used to sort out those molecules from a drug library that interfere with general cellular processes (i.e. replication). By contrast, the system is suitable to identify compounds that specifically interfere with the signaling function of c-di-AMP.

Figure 1 A: 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 RNA polymerase can initiate transcription, the gfp reporter gene is expressed and cells synthesizing GFP become fluorescent.

Figure 1 B: 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.

Reference

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