Team:Goettingen/Team/Array

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*[[Team:Goettingen/Project|Our Project]]
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**[[Team:Goettingen/Project/OurProject|Overview]]
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**[[Team:Goettingen/Team/Reporter|Reporter Team]]
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**<span style="color:#4a7ebb">Array Team</span>
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**[[Team:Goettingen/Team/DAC|DAC Team]]
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===Array Team===
===Array Team===
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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.
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<p> The genome of the Gram-positive model bacterium <i>Bacillus subtilis</i> contains the <i>disA, cdaA</i>, and <i>cdaS</i> genes encoding the three enzymatically active adenylate cyclases DisA, CdaA, and CdaS (Mehne <i>et al.</i> 2013). By using the microarray technique we would like to analyze how many genes are regulated by c-di-AMP in <i>B. subtilis</i>. We are not only interested in the impact of c-di-AMP on the transcriptome, we also hope to identify novel regulatory elements (i.e. riboswitches) and also transcription factors that bind to the essential signaling molecule. In our experiments we use three different <i>B. subtilis</i> strains. The wild-type strain contains all three diadenylate cyclases. The second strain is a <i>disA</i> mutant strain lacking DisA, which was shown to be involved in DNA metabolism (Witte <i>et al.</i> 2008). This strain should produce less c-di-AMP than the isogenic parent strain. The third strain synthesizes a hyperactive CdaS mutant variant, which produces a lot of c-di-AMP. In our microarray experiments we will compare the transcriptomes of each mutant strain with that of the wild-type strain. Moreover, we want to generate a triple knock-out mutant (<i>ΔdisA, ΔcdaA, ΔcdaS</i>) lacking all diadenylate cyclases. We try to construct this mutant strain by feeding with exogenous c-di-AMP. </p>
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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).
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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>.
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https://static.igem.org/mediawiki/2013/a/a6/Goe-arr-fig-1.png
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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''.''
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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 analyze these results, we did some qRT-PCR analysis back in Göttingen (Fig. 2).
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https://static.igem.org/mediawiki/2013/e/e1/Goe-arr-fig-2.png
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''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.''
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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.
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<p>Reference:</p>
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<p> <strong>References</strong></p>
1. Mehne <i>et al.</i> (2013) Cyclic di-AMP homeostasis in <i>Bacillus subtilis</i>: both lack and high level accumulation of the nucleotide are detrimental for cell growth. <i>J. Biol. Chem.</i> 288:2004-2017.
1. Mehne <i>et al.</i> (2013) Cyclic di-AMP homeostasis in <i>Bacillus subtilis</i>: both lack and high level accumulation of the nucleotide are detrimental for cell growth. <i>J. Biol. Chem.</i> 288:2004-2017.
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2. Witte <i>et al.</i> (2008) Structural Biochemistry of a Bacterial Checkpoint Protein Reveals Diadenylate Cyclase Activity Regulated by DNA Recombination Intermediates. <i>Mol. Cell.</i> 30:167-178.
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2. Witte <i>et al.</i> (2008) Structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity regulated by DNA recombination intermediates. <i>Mol. Cell.</i> 30:167-178.
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Latest revision as of 13:57, 4 October 2013





The beast and its Achilles heel:

 A novel target to fight multi-resistant pathogenic bacteria



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 transcriptomes of the wild-type B. subtilis strain and a mutant strain, which produces a lot of c-di-AMP (Fig. 1).

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 B. subtilis.

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 analyze these results, we did some qRT-PCR analysis back in Göttingen (Fig. 2).

Goe-arr-fig-2.png

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 analysis 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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