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 upstream its open reading frame, 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 homeostasis 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. Therefore we are able to test the system by providing the E.coli exogenous 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 c-di-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.

We modified, improved and created several biobricks during the construct of our reporter system. To know more, please go to our subteam page and parts page.

Diadenylate cyclase

As the homeostasis of c-di-AMP is crucial for Listeria monocytogenes (Chelsea E. Witte et al.), a gram-positive human pathogen, we felt very necessary to look closely into the enzyme which produces it, cause it could be a very good target for novel antibiotics. We have chosen the diadenylate cyclase(DAC) from Listera instead of more often used organism Bacillus substilis because the DAC in Bacillus substilis is relative difficult to purify, thus to characterize.

We first tried to express the whole enzyme in E.coli but we couldn't get any clone after a lot of attempts. The full length protein is possibly toxic to E.coli, so we swift our focus from the whole DAC to the catalytic domain of DAC. We attached Strep-tag to the N-terminus of truncated DAC (the catalytic domain) and purified the catalytic domain of DAC with nickel affinity chromatography.

After we get the purified catalytic domain of DAC, we accomplished the kinetic characterization. The results showed that the purified catalytic domain of DAC is still highly functional in vitro. This catalytic domain of DAC is qualified for the construct of screening system, as the final goal of our whole project. Also our experiment went on to our final step, crystallization and finally the determination of the 3D structure.

With the collaboration with Dr. Achim Dickmanns, we successfully get the protein crystal and with the help of Dr. Piotr Neumann, we managed to determine the 3D structure out of the X-ray diffraction pattern.

We believe with this 3D structure of catalytic domain of DAC from Listeria determined, the pharmaceutical industry could develop possible models for novel antibiotics interfering DAC function, by further digging of its inhibitory or activate sites.

In our progress we also created a new biobrick, the catalytic domain of DAC from Literia. Due to limited time, we weren't be able to purify other DACs from other gram-positive bacteria. We really hope for someone or team could carry on to build a biobrick library of all identified DACs.

To know more, please go to our subteam page and parts page.

Mr.Green Coli with his full Bio Gear

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