Team:Northwestern/Project/Introduction

From 2011.igem.org

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Introduction
 
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[[Image:NU_project_introduction_fig1.png|right|frame|'''Figure 1:''' Cell-to-cell signaling system in ''P. aeruginosa'' [http://www.cdc.gov/ncidod/eid/vol4no4/vandelden.htm]]]
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The Basic Idea</DIV>
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Virulence factors produced by ''Pseudomonas aeruginosa'' are controlled by a natural regulatory, hierarchical network consisting of two main systems: the las system and the rhl system. The main components of these systems are the receptor proteins (lasR and rhlR) and the autoinducers (3-oxo-C12-HSL and C4-HSL). The formation of a lasR/3-oxo-C12-HSL complex activates the transcription of several genes, notably rhlR as well as others responsible for biofilm differentiation. The binding of the C4-HSL autoinducer to rhlR further activates genes including the rhlAB operon, which encodes enzymes for the production of rhamnolipids that modulate the motility of ''P. aeruginosa''. Both lasR and rhlR are subject to positive feedback autoregulation through autoinducers. Figure 1 details the existing quorum sensing system inherent to ''P. aeruginosa''.
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Cell signaling and the quorum sensing is mediated by concentrations of the autoinducers PAI-1 and PAI-2, which is why their mere presence indicates existence of P. aeruginosa [12]. The production of PAI-1 and PAI-2 are specific to only the las and the rhl systems respectively [13]. Transcriptional activation of the target gene can be amplified up to 1000-fold due to the binding of the R-protein-autoinducer dimer complex and the induced promoter [1]. Therefore, detecting the presence of P. aeruginosa can be reliant upon the detection of the autoinducers PAI-1 and PAI-2. Our detection system uses the specificity of the autoinducers as a definitive means of detecting P. aeruginosa. This approach focuses primarily on the robust detection of P. aeruginosa, without affecting the pathogen itself.
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Our project goal is to utilize several genes from this existing system, specifically to introduce las and rhl receptors into ''E. coli'' in order to sense the presence of 3-oxo-C12-HSL and/or C4-HSL, thereby detecting ''P. Aeruginosa''. Figure 2 illustrates the genetic circuit that we are attempting to incorporate into ''E. coli''.  
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<caption align="bottom"></html>'''Figure 1:''' Activation of the induced promoters of ''P. aeruginosa'' Las and Rhl sequences by autoinducer/R-protein complexes (transcriptional regulation)<html></caption>
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<tr><td><img src="https://static.igem.org/mediawiki/2011/1/13/Induced_outline.jpg" style="opacity:1;filter:alpha(opacity=100);" width="600px" height="230px" alt="fig1"/ border="0"></td></tr>
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<DIV style="font-size:20px">The Detection System</DIV>
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The detection system incorporates the use of the LasR and RhlR as the cognate R-proteins of the autoinducers PAI-1 and PAI-2. As strongly supported by experimental evidence, LasR/PAI-1 and RhlR and PAI-2 protein complexes act primarily as transcriptional factors [11]. Therefore, our construct will be equipped with inducible promoters extracted from the genome of P. aeruginosa as indicated in Figure 2. These promoters are complementary to the dimer protein complexes, LasP for LasR/PAI-1 and RhlP for RhlR/PAI-2. The sole purpose of the inducible promoters is to facilitate the production of a reporting protein such as GFP and/or RFP as a means of P. aeruginosa detection.
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<caption align="bottom"></html>'''Figure 2:''' Coupling the induced promoters of ''P. aeruginosa'' with reporting constructs (inserts1 and 2)<html></caption>
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<tr><td><img src="https://static.igem.org/mediawiki/2011/1/10/Induced_new.jpg" style="opacity:1;filter:alpha(opacity=100);" width="600px" height="140px" alt="fig1"/ border="0"></td></tr>
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<DIV style="font-size:20px">The Project Goal</DIV><DIV style="font:15px Helvetica;">
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In our construct, the lasR and rhlR gene will be paired with an upstream high efficiency constitutive promoter and ribosome binding site (RBS). Over time, constitutively encoded LasR and RhlR proteins will saturate the cell. Cell signaling autoinducers – PAI-1 and PAI-2 – will be the rate limiting reagents of the reporting genes’ biochemical induction process. The rate of binding of the autoinducers to their cognate R-proteins outpaces the rate of autoinducer degradation. Therefore upon the release of PAI-1 and PAI-2, our engineered E. coli will immediately commence the reporting process.
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'''Figure 2:''' Genetic circuit for ''E. coli'', incorporating several elements from ''P. aeruginosa''.
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Revision as of 07:54, 25 September 2011

RETURN TO IGEM 2010



The Basic Idea

Cell signaling and the quorum sensing is mediated by concentrations of the autoinducers PAI-1 and PAI-2, which is why their mere presence indicates existence of P. aeruginosa [12]. The production of PAI-1 and PAI-2 are specific to only the las and the rhl systems respectively [13]. Transcriptional activation of the target gene can be amplified up to 1000-fold due to the binding of the R-protein-autoinducer dimer complex and the induced promoter [1]. Therefore, detecting the presence of P. aeruginosa can be reliant upon the detection of the autoinducers PAI-1 and PAI-2. Our detection system uses the specificity of the autoinducers as a definitive means of detecting P. aeruginosa. This approach focuses primarily on the robust detection of P. aeruginosa, without affecting the pathogen itself.



Figure 1: Activation of the induced promoters of P. aeruginosa Las and Rhl sequences by autoinducer/R-protein complexes (transcriptional regulation)
fig1


The Detection System

The detection system incorporates the use of the LasR and RhlR as the cognate R-proteins of the autoinducers PAI-1 and PAI-2. As strongly supported by experimental evidence, LasR/PAI-1 and RhlR and PAI-2 protein complexes act primarily as transcriptional factors [11]. Therefore, our construct will be equipped with inducible promoters extracted from the genome of P. aeruginosa as indicated in Figure 2. These promoters are complementary to the dimer protein complexes, LasP for LasR/PAI-1 and RhlP for RhlR/PAI-2. The sole purpose of the inducible promoters is to facilitate the production of a reporting protein such as GFP and/or RFP as a means of P. aeruginosa detection.



Figure 2: Coupling the induced promoters of P. aeruginosa with reporting constructs (inserts1 and 2)
fig1


The Project Goal

In our construct, the lasR and rhlR gene will be paired with an upstream high efficiency constitutive promoter and ribosome binding site (RBS). Over time, constitutively encoded LasR and RhlR proteins will saturate the cell. Cell signaling autoinducers – PAI-1 and PAI-2 – will be the rate limiting reagents of the reporting genes’ biochemical induction process. The rate of binding of the autoinducers to their cognate R-proteins outpaces the rate of autoinducer degradation. Therefore upon the release of PAI-1 and PAI-2, our engineered E. coli will immediately commence the reporting process.

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