Team:Goettingen/Team/Array

From 2013.igem.org

(Difference between revisions)
(→ )
(→Array Team)
Line 40: Line 40:
===Array Team===
===Array Team===
-
<html>
+
With global target analyses, 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''.
-
<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). At least one of the three enzymes is needed for growth of the bacteria. 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, a protein that 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 (Mehne <i>et al.</i> 2013). 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 the cells with exogenous c-di-AMP that is commercially available. </p>
+
 
-
</html>
+
Our goal was to look for genes, whose expression level is affected by the level of c-di-AMP. Therefore we compared the wildtypewith 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.
 +
 
 +
https://static.igem.org/mediawiki/2013/a/a6/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 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).
 +
 
 +
https://static.igem.org/mediawiki/2013/e/e1/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 ofydaOis supposedly linked to the c-di-AMP levels inside the cell.
 +
 
 +
 
 +
 
 +
 
<p> <strong>References</strong></p>
<p> <strong>References</strong></p>
Line 48: Line 66:
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.
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.
 +
===&nbsp; ===
===&nbsp; ===
<html>
<html>

Revision as of 10:21, 1 October 2013





The beast and its Achilles heel:

 A novel target to fight multi-resistant pathogenic bacteria



Array Team

With global target analyses, 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 wildtypewith 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 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).

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 ofydaOis 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.

 

Previous Next


Retrieved from "http://2013.igem.org/Team:Goettingen/Team/Array"