In order to facilitate the development of new antibiotics, we want to build an “Antibiotic Detector” that can identify substances influencing the homeostasis of cyclic-di-AMP (c-di-AMP). c-di-AMP is an essential signal nucleotide in most Gram-positive bacteria. It is not found in humas and several Gram-negative bacteria lack this compound, as well, including E. coli. For more information see Our Project overview.
For constructing a c-di-AMP detector, we need sensors for c-di-AMP and in order to visualize the output of the sensors, we need a reporter.
Recently, in Mycobacterium smegmatis, a transcriptional repressor (DarR) was identified. It can bind to a specific DNA sequence, called the DarR operator. The binding efficiency of DarR is strongly enhanced by c-di-AMP. Since DarR is a c-di-AMP sensor, we intended to use it for our reporter system. We cloned DarR into pSB1C3 ([http://parts.igem.org/Part:BBa_K1045001 BBa_K1045001]) and also constructed the operator as a Biobrick ([http://parts.igem.org/Part:BBa_K1045000 BBa_K1045000]). We placed the DarR operator between a strong promoter ([http://parts.igem.org/Part:BBa_J23110 BBa_J23110]) and the GFP generator [http://parts.igem.org/Part:BBa_E0240 BBa_E0240]. In the very same plasmid but oriented in the opposite direction, we assembled a DarR expression unit. DarR expression was driven by a weak promoter based on [http://parts.igem.org/Part:BBa_J23117 BBa_J23117] and terminated by [http://parts.igem.org/Part:BBa_K1045009 BBa_K1045009]. This terminator is based on [http://parts.igem.org/Part:BBa_B0015 BBa_B0015], which is part of [http://parts.igem.org/Part:BBa_E0240 BBa_E0240], as well, and should ensure that the transcription of DarR and GFP did not influence each other. The RBS [http://parts.igem.org/Part:BBa_K1045010 BBa_K1045010] (derived from [http://parts.igem.org/Part:BBa_B0034 BBa_B0034]) was used as an RBS for DarR mRNA translation. Assembly of all those parts (Fig. 1.1) required a complex cloning process which finally resulted in part [http://parts.igem.org/Part:BBa_K1045017 BBa_K1045017], the DarR reporter system (Fig. 1.2). E. coli was then transformed with this construct to obtain the in vivo screening system for antibiotics directed against c-di-AMP.
Ideally, this c-di-AMP-sensing system works in the following way (Fig. 1.3): Without c-di-AMP, DarR is not bound to the DarR operator, so GFP is expressed resulting in green fluorescing cells. With c-di-AMP, DarR can bind to its binding sequence, repressing gfp transcription, leading to non-fluorescent cells. In the same way, the system might react to compounds similar to c-di-AMP. It can therefore be used to screen for compounds similar to c-di-AMP. It can be used in a large throughput scale, as well. This way, the DarR reporter system will make it possible to find inhibitors and competitors which might be used as antibiotics.
Reference:
1. Zhang et.al.(2013)DarR, a TetR-like Transcriptional Factor, Is a Cyclic Di-AMP-responsive Repressor in Mycobacterium smegmatis, J. Biol. Chem. 288:3085–3096