Team:Goettingen/Team/Array

From 2013.igem.org

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''Fig.1: C-di-AMP concentrations determined via liquid chromatography-coupled tandem mass spectrometry method by our collaboration partner from the Hannover Medical School. In blue the c-di-AMP amount in low phosphate medium is shown, in red the c-di-AMP amount in high phosphate medium. We compared the wildtype and a hyperactive strain (1344) of ''Bacillus''.''
''Fig.1: C-di-AMP concentrations determined via liquid chromatography-coupled tandem mass spectrometry method by our collaboration partner from the Hannover Medical School. In blue the c-di-AMP amount in low phosphate medium is shown, in red the c-di-AMP amount in high phosphate medium. We compared the wildtype and a hyperactive strain (1344) of ''Bacillus''.''
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The microarray analyses in Groningen gave us a first glimpse on all the genes, which are affected by the c-di-AMP level. We were especially interested in ''ydaO''. From a poster of another workgroup we knew that ''ydaO'' is connected to an upstream c-di-AMP sensing riboswitch. This means that in the presence of c-di-AMP the riboswitch will assemble and prohibit the expression of ''ydaO''. With no c-di-AMP the riboswitch cannot be established and ''ydaO'' will be expressed. Therefore, ''ydaO'' was a perfect candidate for us to build another reporter around it. As the microarray analyses revealed, the expression of ''ydaO'' was indeed affected by c-di-AMP levels. To further analyse these results, we did some qRT-PCR analyses back in Göttingen (Fig. 2).
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The microarray analysis in Groningen gave us a first glimpse on all the genes, which are affected by the c-di-AMP level. We were especially interested in ''ydaO''. From a poster of another workgroup we knew that ''ydaO'' is connected to an upstream c-di-AMP sensing riboswitch. This means that in the presence of c-di-AMP the riboswitch will assemble and prohibit the expression of ''ydaO''. Without c-di-AMP the riboswitch cannot be established and ''ydaO'' will be expressed. Therefore, ''ydaO'' was a perfect candidate for us to build another reporter around it. As the microarray analysis revealed, the expression of ''ydaO'' was indeed affected by c-di-AMP levels. To further analyse these results, we did some qRT-PCR analysis back in Göttingen (Fig. 2).
https://static.igem.org/mediawiki/2013/e/e1/Goe-arr-fig-2.png
https://static.igem.org/mediawiki/2013/e/e1/Goe-arr-fig-2.png
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''Fig.2: qRT-PCR analyses of c-di-AMP affected ''ydaO'' and a of control gene. The analyses revealed that in comparison to the wild type ydaO is upregulated with low c-di-AMP levels.''
''Fig.2: qRT-PCR analyses of c-di-AMP affected ''ydaO'' and a of control gene. The analyses revealed that in comparison to the wild type ydaO is upregulated with low c-di-AMP levels.''
-
Our qRT-PCR analyses showed that ydaO is upregulated with low c-di-AMP levels. This means that the expression ofydaOis supposedly linked to the c-di-AMP levels inside the cell.
+
Our qRT-PCR analyses showed that ydaO is upregulated with low c-di-AMP levels. This means that the expression of ''ydaO'' is supposedly linked to the c-di-AMP levels inside the cell.

Revision as of 09:34, 2 October 2013





The beast and its Achilles heel:

 A novel target to fight multi-resistant pathogenic bacteria



Array Team

With global target analysis, which were performed in collaboration with the iGEM Groningen Team, we were interested in identifying novel c-di-AMP regulatory elements that control gene expression in Bacillus subtilis.

Our goal was to look for genes, whose expression level is affected by the level of c-di-AMP. Therefore we compared the wildtype with a hyperactive strain of Bacillus, which produces more c-di-AMP (Fig. 1).

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.

Goe-arr-fig-1.png

Fig.1: C-di-AMP concentrations determined via liquid chromatography-coupled tandem mass spectrometry method by our collaboration partner from the Hannover Medical School. In blue the c-di-AMP amount in low phosphate medium is shown, in red the c-di-AMP amount in high phosphate medium. We compared the wildtype and a hyperactive strain (1344) of Bacillus.

The microarray analysis in Groningen gave us a first glimpse on all the genes, which are affected by the c-di-AMP level. We were especially interested in ydaO. From a poster of another workgroup we knew that ydaO is connected to an upstream c-di-AMP sensing riboswitch. This means that in the presence of c-di-AMP the riboswitch will assemble and prohibit the expression of ydaO. Without c-di-AMP the riboswitch cannot be established and ydaO will be expressed. Therefore, ydaO was a perfect candidate for us to build another reporter around it. As the microarray analysis revealed, the expression of ydaO was indeed affected by c-di-AMP levels. To further analyse these results, we did some qRT-PCR analysis back in Göttingen (Fig. 2).

Goe-arr-fig-2.png

Fig.2: qRT-PCR analyses of c-di-AMP affected ydaO and a of control gene. The analyses revealed that in comparison to the wild type ydaO is upregulated with low c-di-AMP levels.

Our qRT-PCR analyses showed that ydaO is upregulated with low c-di-AMP levels. This means that the expression of ydaO is supposedly linked to the c-di-AMP levels inside the cell.



References

1. Mehne et al. (2013) Cyclic di-AMP homeostasis in Bacillus subtilis: both lack and high level accumulation of the nucleotide are detrimental for cell growth. J. Biol. Chem. 288:2004-2017.

2. Witte et al. (2008) Structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity regulated by DNA recombination intermediates. Mol. Cell. 30:167-178.

 

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