Team:Goettingen/Project/OurProject

Our Project

Our project is aimed at finding a way to fight against multi-resistant bacteria by targeting c-di-AMP. We made three different approaches.

We built two reporter system, with which we are able to visualize the level of c-di-AMP. (accomplished by Reporter Team).

we also searched for the genes in Bacillus substilis, whose expression level is effected by the level of c-di-AMP. We found ydaO and identified a Ribo-Switch which responds to c-di-AMP. We used the ydaO Riboswitch directly in our second reporter system. (accomplished by Array Team).

Last but not least, we looked into the diadenylate cyclase(DAC) from Listeria monocytogenes. We successfully expressed tagged truncated DAC(catalytic domain) in E.coli and purified it. We tested its kinetic characteristics and crystallized it. In the end, we are able to determine the STRUCTURE. (accomplished by DAC Team)

Reporter systems

For the Reporter team, our final goal is to build a screening system, which allows quick identification and characterization of substances which are able to disturb c-di-AMP homeostasis in pathogenic bacteria. We believe the accomplished screening system will be a great help for pharmaceutical industry worldwide in finding new and more effective antibiotics against Gram-positive pathogens, for which c-di-AMP homestasis is crucial. To accomplish that goal, we first need a reporter system of c-di-AMP, with which we can visualize level of c-di-AMP.

We used a few existing Biobricks and also created new ones to build up our system. At first, we attempted to construct a reporter system which is controlled by DarR, a transcriptional inhibitor identified in Mycobacterium smegmatis. This reporter system consists of three parts, namely a constitutively active promoter, the operator sequence DarR binds and a reporter gene cassette. c-di-AMP is able to stimulate the binding of DarR and its operator, acting as a co-inhibitor. Therefore the level of c-di-AMP can be visualized by the fluorescence of GFP: the higher the c-di-AMP level is, the lower the GFP fluorescence becomes. The reporter system is transformed into E.coli, which produces no endogens c-di-AMP, but is able to take up c-di-AMP from the environment. Therefore we are able to test the system by feeding the E.coli c-di-AMP.

The second reporter system is based on the result of our Array Team. They found out the gene ydaO, whose expression level is effected by the level of c-di-AMP. When the cdi-AMP level is low, the ydaO expression is up-regulated. We are able to identify a Riboswitch upstream the ydaO open reading frame and used it in our second reporter system. The ydaO Riboswitch has two states: "ON" and "OFF". The switch between the two states depends on the presence of c-di-AMP: basically, when c-di-AMP is there, the Riboswitch is "OFF" and when there is no c-di-AMP, the Riboswitch is "ON". We cloned a reporter gene cassette CFP downstream the native promoter + ydaO Riboswitch. This reporter system should act similarly to our first reporter system: when there is c-di-AMP, no signal, but when there is no c-di-AMP, there will be a fluorescence signal.

Diadeylate cyclase

Originating from the same organism, B. subtilis, we want to take thediadenylatecyclase (DacA)to be able to produce endogenous c-di-AMP. This might be necessary, depending on the uptake of exogenous c-di-AMP into E.coli. (model of DacA)

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Furthermore was it an aim of our project to identify the structure of the DacA domain using crystalography. Knowing the structure of the protein, which is responsible for the production of c-di-AMP in its host, is supposed to help in the identification or even synthesis of possible antibiotics.

There are many advantages in using E.coli as a host for our reporter system. Besides its low prize and ease to handle, E.coli itself does not use or produce c-di-AMP. Therefore, bringing it into the organism and inhibiting it again does not influence the growth of E.coli or even kill it. The only detectable effect will be shown by our reporter system.

We are confident that our screening system will facilitate the identification of novel antibacterial substances because any change in the activity of the c-di-AMP-dependent promoter-reporter gene fusion, either by inhibition of c-di-AMP synthesis or by activation of DNA-binding activity of the transcription factor will indicate perturbation of c-di-AMP homeostasis.

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