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Team:Goettingen/Team/Array
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===Array Team=== | ===Array Team=== | ||
| - | + | We were interested in identifying novel c-di-AMP regulatory elements that control gene expression in ''Bacillus subtilis''. In collaboration with the iGEM Groningen Team, we performed global target analysis. | |
| - | Our goal was to look for genes, whose expression level is affected by the level of c-di-AMP. Therefore we compared the | + | Our goal was to look for genes, whose expression level is affected by the level of c-di-AMP. Therefore we compared the transcriptomes of the wild-type <i>B. subtilis</i> strain and a mutant strain, which produces a lot of c-di-AMP (Fig. 1). |
| - | By doing so, we are able to | + | By doing so, we are able to (i) find other pathways that respond to c-di-AMP and therefore can be used to construct a reporter system, and (ii) we can shed light on the signaling network of c-di-AMP in <i>B. subtilis</i>. |
https://static.igem.org/mediawiki/2013/a/a6/Goe-arr-fig-1.png | https://static.igem.org/mediawiki/2013/a/a6/Goe-arr-fig-1.png | ||
| - | ''Fig.1: | + | ''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 | + | 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 analyze 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 | ||
| - | ''Fig.2: qRT-PCR | + | ''Fig.2: qRT-PCR analysis of c-di-AMP affected ''ydaO'' and a of control gene. The analysis revealed that in comparison to the wild type ydaO is upregulated with low c-di-AMP levels.'' |
| - | Our qRT-PCR | + | Our qRT-PCR analysis showed that <i>ydaO</i> 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. |
Latest revision as of 13:57, 4 October 2013
