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 systems, 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.

Microarray

We array team focused mainly on the genes, whose expression level is regulated by c-di-AMP. Therefore we compared the transcriptomic data of wild type with those of a hyperactive strain of Bacillus, which produces more c-di-AMP.

By doing so, we are able to: first, find other compartments which also respond to c-di-AMP and therefore can be used to construct our reporter system, second we can shed light on the signaling web of c-di-AMP.

With the collaboration of iGEM Team Groningen, we accomplished the microarray analysis, which has given us a first glimpse of on all genes which could be regulated by c-di-AMP. Among those genes, ydaO caught most our attention. ydaO motif is reported to be an ATP-sensing riboswitch in Bacillus substilis (Peter Y Watson & Martha J Fedor, 2012). It's a bit surprising that ydaO appeared on this map. We confirmed the array data with qRT-PCR. And the results consisted with array data. Therefore we believe we identified another genetic element that responds to c-di-AMP and we used it directly to build our secondary reporter system.

To know more about our work, please visit subteam page and parts page.

Diadenylate cyclase

As the homeostasis of c-di-AMP is very important for Listeria monocytogenes (Chelsea E. Witte et al.), a gram-positive human pathogen, We are convinced that the DAC is a very promising target for the development of highly specific antibiotic substances which exclusively act on Gram-positive bacteria and are not harmful for Gram-negative ones, including the gut bacterium Escherichia coli. Therefore, we aimed to reveal the protein structure of a not yet characterized diadenylate cyclase domain.

First we tried to express the complete CdaA protein of L. monocytogenes in E.coli. Unfortunately, we couldn't get any clone after several attempts. The full length protein might be toxic expressed in E.coli, so we focused on a truncated version of the whole protein excluding the trans-membrane domains. The 173 amino acid protein fragment (100 – 273 of CdaA) harbors exclusively the catalytic domain of CdaA, also referred to as DacA. We attached an N-terminal Strep-tag to the cyclase domain and expressed the protein driven by a T7-promoter. HPLC-MS/MS measurements determined the presence of c-di-AMP and, thus, the activity of the catalytic domain in also in the Gram-negative bacterium E.coli in vivo.

Excluding the trans-membrane domains, the protein localizes to the cytoplasm and can be purified using streptavidin purification columns. After we purified the DAC protein, we wanted to proof the functionality also in vitro. The results showed that the purified catalytic domain of CdaA is still functional in vitro in proper conditions. Thus, the truncated diadenylate cyclase domain is qualified to construct of a screening system with the ectopic production of c-di-AMP in E.coli, as a further goal of our whole project. Also our experiments went on to our final step, crystallization and, finally, the determination of the 3D protein structure.

In collaboration with Dr. Achim Dickmanns, we successfully obtained protein crystals and with the help of Dr. Piotr Neumann, we managed to determine the 3D structure from the X-ray diffraction pattern.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 the 3D structure of the diadenylate cyclase domainofListeria, the pharmaceutical industry could develop possible models for the devolpment of novel antibiotics interfering with DAC function.

In our progress we also created a new biobrick, to know more, please go to our subteam page and parts page.

Mr.Green Coli with his full Bio Gear

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