Team:Goettingen/Team/Reporter

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**<span style="color:#4a7ebb">Reporter Team</span>
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**[[Team:Goettingen/Team/Array|Array Team]]
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===Reporter Team===
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<p>The <i>in vivo</i> activity of a transcriptional regulator can be easily detected by monitoring the activity of a promoter that is fused to a reporter gene such as the green fluorescent protein (GFP). Recently, in <i>Mycobacterium smegmatis</i>, the transcriptional repressor DarR was identified (Zhang <i>et al.</i> 2013). DarR is capable of binding to a specific DNA sequence upon association with c-di-AMP. This led us to the idea of developing a powerful screening system to facilitate the identification of potential drugs interfering with c-di-AMP biofunction. While many Gram-positive bacteria (also pathogenic bacteria that cause severe human diseases) require c-di-AMP for growth, c-di-AMP is not synthesized by the Gram-negative bacterium <i>Escherichia coli</i>. Therefore, we intend to build a reporter system that is composed of existing and of novel biobricks that will be developed during the project (Figure 1). As <i>E. coli</i> does not require c-di-AMP for growth the screening system can be used to sort out those molecules from a drug library that interfere with general cellular processes (i.e. replication). By contrast, the system is suitable to identify compounds that specifically interfere with the signaling function of c-di-AMP. </p>
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<p><b>Figure 1 A:</b> In the absence of c-di-AMP, DarR cannot bind to its binding site (operator) that is located downstream of the promoter. As a consequence RNA polymerase can initiate transcription, the <i>gfp</i> reporter gene is expressed and cells synthesizing GFP become fluorescent.</p>
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<p>The activity of transcriptional regulators can be easily monitored by expressing a reporter gene downstream of a promoter, containing their binding site. Recently, in Mycobacterium smegmatis, a transcriptional repressor (DarR) was identified that is able to bind to a specific DNA sequence upon association with c-di-AMP. This led us to the idea of developing a screening system for potential drugs interfering with c-di-AMP biofunction. While many gram-positive bacteria require c-di-AMP for their growth, this molecule is not synthesized by the gram-negative bacterium E. coli. Thus, we intend to build a reporter system consisting of the iGEM biobricks in E. coli (Figure 1).</p>
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<p><b>Figure 1 B: </b> In the presence of c-di-AMP, the signaling molecule stimulates DNA-binding activity of DarR and the c-di-AMP-DarR complex prevents transcription initiation by RNA polymerase. The <i>E. coli</i> cells do not produce GFP and are therefore non-fluorescent.</p></div>
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<div style="width:70%" class="cen"><b>Figure 1a:</b> When there is no c-di-AMP present, the repressor gene DarR cannot bind to its binding site(operator sequence). The transciption starts and leads to the expression of the reporter gene. This reporter gene could be GFP, or other widely used reporter gene.</div>
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<p><strong>Reference</strong></p>
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1. <a href="http://www.jbc.org/content/288/5/3085" style="color:#303030" >Zhang <i>et al.</i> (2013) DarR, a TetR-like transcriptional factor, Is a cyclic di-AMP-responsive repressor in <i>Mycobacterium smegmatis, J. Biol. Chem. </i> 288:3085–3096</a>
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<b>Figure 1b: </b> When there is c-di-AMP present, the c-di-AMP can act as ligand of the repressor gene DarR, activating tis function. DarR binds to its binding site, blocking the RNA polymerase, and therefore disrupt transcription. Then we are supposed to see no signal or reduced signal.</div>
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<p>Reference:</p>
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1. <a href="http://www.jbc.org/content/288/5/3085" style="color:#303030" >Zhang <i>et.</i>al.(2013)DarR, a TetR-like Transcriptional Factor, Is a Cyclic Di-AMP-responsive Repressor in <i>Mycobacterium smegmatis, J. Biol. Chem. </i> 288:3085–3096</a>
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Revision as of 09:08, 1 October 2013





The beast and its Achilles heel:

 A novel target to fight multi-resistant pathogenic bacteria


Reporter Team



The activity of transcriptional regulators can be easily monitored by expressing a reporter gene downstream of a promoter, containing their binding site. Recently, in Mycobacterium smegmatis, a transcriptional repressor (DarR) was identified that is able to bind to a specific DNA sequence upon association with c-di-AMP. This led us to the idea of developing a screening system for potential drugs interfering with c-di-AMP biofunction. While many gram-positive bacteria require c-di-AMP for their growth, this molecule is not synthesized by the gram-negative bacterium E. coli. Thus, we intend to build a reporter system consisting of the iGEM biobricks in E. coli (Figure 1).

Figure 1a: When there is no c-di-AMP present, the repressor gene DarR cannot bind to its binding site(operator sequence). The transciption starts and leads to the expression of the reporter gene. This reporter gene could be GFP, or other widely used reporter gene.

Figure 1b: When there is c-di-AMP present, the c-di-AMP can act as ligand of the repressor gene DarR, activating tis function. DarR binds to its binding site, blocking the RNA polymerase, and therefore disrupt transcription. Then we are supposed to see no signal or reduced signal.

Reference:

1. Zhang et.al.(2013)DarR, a TetR-like Transcriptional Factor, Is a Cyclic Di-AMP-responsive Repressor in Mycobacterium smegmatis, J. Biol. Chem. 288:3085–3096

 

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