Team:Goettingen/Project/OurProject
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==Microarray== | ==Microarray== | ||
- | We | + | We 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. | 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. | ||
- | + | In the collaboration with iGEM Team Groningen, we accomplished the microarray analysis, which has given us a first glimpse 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 ''ydaO'' motif is reported to be a c-di-ATP-sensing Riboswitch in ''Bacillus substilis'' (unpublished data). 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 directly used it to build our second reporter system. | |
To know more about our work, please visit [[Team:Goettingen/Team/Array|subteam page]] and [[Team:Goettingen/Parts|parts page]]. | To know more about our work, please visit [[Team:Goettingen/Team/Array|subteam page]] and [[Team:Goettingen/Parts|parts page]]. | ||
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==Diadenylate cyclase== | ==Diadenylate cyclase== | ||
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- | <p>As the homeostasis of c-di-AMP is very important for <i>Listeria monocytogenes</i> (Witte <i>et al.</i>, 2013), a Gram-positive human pathogen, | + | <p>As the homeostasis of c-di-AMP is very important for <i>Listeria monocytogenes</i> (Witte <i>et al.</i>, 2013), 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 to Gram-negative ones, including the gut bacterium <i>Escherichia coli</i>. Therefore, we aimed to reveal the protein structure of a not yet characterized diadenylate cyclase domain. </p> |
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- | 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 | + | 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 | + | 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 prove 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 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 [[Team:Goettingen/NoteBook/Acknowlegement|Dr. Achim Dickmanns]], we successfully obtained protein crystals and with the help of [[Team:Goettingen/NoteBook/Acknowlegement|Dr. Piotr Neumann]], we managed to determine the 3D structure from | + | In collaboration with [[Team:Goettingen/NoteBook/Acknowlegement|Dr. Achim Dickmanns]], we successfully obtained protein crystals and with the help of [[Team:Goettingen/NoteBook/Acknowlegement|Dr. Piotr Neumann]], we managed to determine the 3D structure from the X-ray diffraction pattern. |
- | We believe | + | We believe that the 3D structure of the diadenylate cyclase domain of ''Listeria'' can help pharmaceutical industry in developing novel antibiotics that interfere with DAC function. |
- | In our progress we also created a new | + | In our progress we also created a new BioBrick, to know more, please go to our [[Team:Goettingen/Team/DAC|subteam page]] and [[Team:Goettingen/Parts|parts page]]. |
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Latest revision as of 13:51, 4 October 2013