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 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)

Last but not least, we 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).

Using given Biobricks and by creating new ones, we first wanted to construct a promoter-reporter fusion system which is under the control of DarR, an operator derived from Mycobacterium smegmatis. Only in the presents of c-di-AMP will this operator able to inhibit the transcription of the reporter gene GFP. The operator binding sequence will be placed between a constitutively active and the reporter gene. The activity of this reporter is rather to detect, since it emits green light if not inhibited. Via the addition of exogenous c-di-AMP, we will have to evaluate whether it is able to inhibit our promoter-reporter system.

By accident we stumbled upon the discovery of a new Ribo-Switch, which is also under the control of c-di-AMP. This Ribo-Switch, called YdaO, was discovered in Bacillus subitilis.With this, we figured, we would have a second way to construct a reporter system. (Green Coli with a SWITCH in his hand)

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)

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.

Mr.Green Coli with his full Bio Gear

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