From 2013.igem.org
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| | ======Our Porject====== | | ======Our Porject====== |
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| - | <h3>Our project: </h3>
| + | <p>Our project is aimed at the development of a simple screening system, which allows the rapid identification and characterization of substances that disturb c-di-AMP homeostasis in pathogenic bacteria. By developing this system, we believe we can help scientist and pharmaceutical companies worldwide to find new and more effective antibiotics against Gram-positive pathogens, for which c-di-AMP is essential. The screening system will be established in the non- pathogenic bacterium <i>E. coli.</i> </p> |
| - | <img src="https://static.igem.org/mediawiki/2013/e/eb/Fig1.png" style="float:right;width:50%" />
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| - | <p>Our project is aimed at the development of a simple screening system, which allows the rapid identification and characterization of substances that disturb c-di-AMP homeostasis in pathogenic bacteria. </p> | + | |
| - | <p> The principle of how such a screening system could look like is illustrated in Figure 1. The screening system will be established in the non-pathogenic bacterium E. coli. </p>
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| - | <p> First, we want to construct a promoter-reporter gene fusion which allows us to monitor the activity of a transcription factor that only binds to a specific DNA sequence (operator) in the presence of c-di-AMP. The operator sequence will be placed between a constitutively active promoter and a reporter gene, such as lacZ and gfp encoding the -galactosidase and the green-fluorescent protein (GFP), respectively. For details,please click <a href="/Team:Goettingen/Team/Reporters" class="moreinfo">Reporter Team</a></p>
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| - | <p> The activity of either of the two proteins is very easy to detect. Then, we will evaluate whether binding of the transcription factor and thus inhibition of the promoter-reporter gene fusion can be controlled by exogenous c-di-AMP. </p>
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| - | <p> Finally, we want to express a diadenylate cyclase from B. subtilis to inhibit the promoter reporter gene fusion by endogenously synthesized c-di-AMP. There are two big advantages of using E. coli as a host for the development of a screening system to identify antibacterial compounds that interfere with c-di-AMP homoeostasis in Gram-positive pathogenic bacteria. </p>
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| - | <p> First, c-di-AMP is not synthesized in E. coli. Thus, compounds that inhibit c-di-AMP synthesis will specifically inhibit growth of Gram-positive bacteria. Second, the use of a nonpathogenic E. coli strain, which is easy to cultivate will keep the costs very low.</p>
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| - | <p> We are confident that our screening system will facilitate the identification of novel antibacterial substances because any change in the activity of the c-di-AMP-dependent promoter-reporter gene fusion, either by inhibition of c-di-AMP synthesis or by activation of DNA-binding activity of the transcription factor will indicate perturbation of c-di-AMP homeostasis. </p>
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Revision as of 13:48, 26 September 2013
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