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The beast and its Achilles heel:
A novel target to fight multi-resistant pathogenic bacteria
Reporter Team
Introduction
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.
DarR reporter system (part BBa_K1045017):
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 (BBa_K1045001) and also constructed the operator as a Biobrick (BBa_K1045000). We placed the DarR operator between a strong promoter (BBa_J23110) and the GFP generator 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 BBa_J23117 and terminated by BBa_K1045009. This terminator is based on BBa_B0015, which is part of BBa_E0240, as well, and should ensure that the transcription of DarR and GFP did not influence each other. The RBS BBa_K1045010 (derived from 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 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. <a href="http://www.jbc.org/content/288/5/3085" style="color:#303030" >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</a>
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