Team:Goettingen/Achievement

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Team: Goettingen

Achievements

We apply for

Best BioBrick Measurement Approach

Best Model

The progress in the development of new antibiotics has been very slow during the last decades, leaving many strains of bacteria untreatable. This problem has been growing ever since the invention of antibiotics but has now become a real threat to millions of people around the world. This Threat we want to tackle with a new approach: We develop a system to test substances on a new target, c-di-AMP.

C-di-AMP was discovered to be an essential signaling molecule in Gram-positive bacteria including the pathogens Streptococcus pneumoniae, Staphylococcus aureus and Listeria monocytogenes. Both loss and overproduction of c-di-AMP have detrimental effects on cell growth, cell wall synthesis and propagation.

Best BioBrick Measurement Approach:

Thus, the diadenylate cyclase (DAC) which catalyses the condensation reaction of 2 ATP molecules to c-di-AMP is the key factor for c-di-AMP homeostasis. We are convinced that the DAC protein is a very promising target for the development of highly specific antibiotic substances which exclusively act on Gram-positive bacteria and are not harming Gram-negative ones, including the gut bacterium Escherichia coli as well as humans. We characterized the DAC in vivo and in vitro. Here, we introduce a truncated but functional DAC, which localizes to the cytosol and can easily be purified. Furthermore, we obtained protein crystals and the protein structure by X-ray diffraction analysis. For the first time ever the 3D structure for this cyclase, which is conserved among all important Gram-positive bacteria, was resolved. This structure will be an important starting point for bioinformatic docking studies (BBa_K1045003)

Best Model Approach:

To test an antibiotic on the new target, we isolated (as mentioned above) the truncated DAC and did activity measurements on it. Using this data, we can now screen thousands of substances for impacts on the function of the DAC (in silico and in vitro screening). Furthermore, we created in vivo reporter systems in E. coli, giving us the ability to sense the amount of c-di-AMP inside the cells, as well as to find possible inhibitors or competitors for c-di-AMP. Last but not least, the combination of the two: An in vivo reporter system with the DAC expressed in the same cell will be the c-di-AMP pathway from Gram-positive bacteria, reconstructed in E. coli. This way, it will be ready to screen for substances lethal to it´s Gram-positive relatives, without harming our host. In consequence, our model will facilitate the search for novel antibiotics hitting a novel target in Gram-positive bacteria.

About our parts

We have two new parts built, namely: [http://partsregistry/Part:BBa_K1045003 BBa_K1045003],[http://partsregistry/Part:BBa_K1045017 BBa_K1045017]

We also improved some parts, one of them is [http://partsregistry/Part:BBa_K1045011 BBa_K1045011]

We did qRT PCR analysis, single cell microscopy and RFP analyses in different growth phases of the original part BBa_J23117. These measurements contribute to a highly increased characterization of this part and three others from it´s promoter family. We also inverted this part, thus improving it and making it usable in an inverted expression unit. This we used in order to express our repressor DarR on the same vector as our reporter system. The improved part submitted to the registry is BBa_K1045011.