Team:Northwestern/Project/Introduction

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<DIV style="font-size:20px">The Basic Idea</DIV>
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<DIV style="font-size:20px">Basic Idea</DIV>
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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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Cell signaling and quorum sensing in ''P. aeruginosa'' is mediated by the autoinducers PAI-1 and PAI-2, so these molecules are excellent indicators that this bacterium is present. <html><sup><A HREF="#1">[1]</A></sup></html>. The production of PAI-1 and PAI-2 are specific to the las and the rhl systems respectively<html><sup><A HREF="#2">[2]</A></sup></html>. Transcriptional activation of target genes can be amplified up to 1000-fold due to the binding of the R-protein autoinducer/dimer complex to its respective target promoter<html><sup><A HREF="#3">[3]</A></sup></html>. Therefore, our detection system uses the specificity of these two autoinducer-response systems as a definitive means of detecting ''P. aeruginosa''.
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<DIV style="font-size:20px">The Detection System</DIV>
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<DIV style="font-size:20px">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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Our detection system incorporates LasR and RhlR receptors (R-proteins) for the autoinducers PAI-1 and PAI-2. As previously reported, LasR/PAI-1 and RhlR/PAI-2 protein complexes act primarily as transcriptional factors<html><sup><A HREF="#4">[4]</A></sup></html>. Therefore, our platform will utilize inducible promoters from the genome of ''P. aeruginosa'' as indicated in Figure 2. These promoters are induced by their respective ligand-bound dimeric R-protein complexes, LasP for LasR/PAI-1 and RhlP for RhlR/PAI-2. In our proof-of-concept system, the inducible promoters drive the expression of reporter proteins, such as GFP or RFP, to report the presence of ''P. aeruginosa'' autoinducer molecules through a readout that is easily quantified. Future implications of this concept could include reporter genes that are detectable by other means, such as a simple visual color-change system.
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<DIV style="font-size:20px">The Project Goal</DIV>
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<DIV style="font-size:20px">Design Considerations</DIV>
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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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In our system, the lasR and rhlR genes are expressed from a high efficiency constitutive promoter and ribosome binding site (RBS). Given this high steady-state expression of LasR and RhlR proteins, we predict that the autoinducers PAI-1 and PAI-2 will be rate-limiting for the induction of the reporter genes. Therefore, reporter gene expression should commence rapidly upon the binding of PAI-1 or PAI-2 to their respective R-protein.
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<html><A NAME="1" href="http://www.ncbi.nlm.nih.gov/pubmed/7494482">[1]</A>  Latifi A, Winson MK, Foglino M, Bycroft Bw, Stewart GS, Lazdunski A, et al. Multiple homologues of LuxR and LuxI control expression of virulence determinants and secondary metabolites through quorum sensing in Pseudomonas aeruginosa PAO1. Mol Microbiol 1995;17:333-43.</html><br>
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<html><A NAME="2" href="http://www.ncbi.nlm.nih.gov/pubmed/10984043">[2]</A>  Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, et al. Complete genome sequence ofPseudomonas aeruginosa PA01, an opportunistic pathogen. Nature. 2000;406:959–964.</html><br>
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<html><A NAME="3" href="http://jb.asm.org/cgi/content/abstract/185/7/2080">[3]</A>  Wagner VE, Bushnell D, Passador L, Brooks AI, Iglewski BH. Microarray analysis ofPseudomonas aeruginosa quorum-sensing regulons: Effects of growth phase and environment. J Bacteriol. 2003;185:2080–2095.</html><br>
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<html><A NAME="4" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC179088/">[4]</A>  Pesci EC, Pearson JP, Seed PC, Iglewski BH. Regulation of las and rhl quorum sensing inPseudomonas aeruginosa. J Bacteriol 1997;179:3127-32.</html><br>
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Latest revision as of 02:10, 29 October 2011

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Basic Idea


Cell signaling and quorum sensing in P. aeruginosa is mediated by the autoinducers PAI-1 and PAI-2, so these molecules are excellent indicators that this bacterium is present. [1]. The production of PAI-1 and PAI-2 are specific to the las and the rhl systems respectively[2]. Transcriptional activation of target genes can be amplified up to 1000-fold due to the binding of the R-protein autoinducer/dimer complex to its respective target promoter[3]. Therefore, our detection system uses the specificity of these two autoinducer-response systems as a definitive means of detecting P. aeruginosa.


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


Detection System


Our detection system incorporates LasR and RhlR receptors (R-proteins) for the autoinducers PAI-1 and PAI-2. As previously reported, LasR/PAI-1 and RhlR/PAI-2 protein complexes act primarily as transcriptional factors[4]. Therefore, our platform will utilize inducible promoters from the genome of P. aeruginosa as indicated in Figure 2. These promoters are induced by their respective ligand-bound dimeric R-protein complexes, LasP for LasR/PAI-1 and RhlP for RhlR/PAI-2. In our proof-of-concept system, the inducible promoters drive the expression of reporter proteins, such as GFP or RFP, to report the presence of P. aeruginosa autoinducer molecules through a readout that is easily quantified. Future implications of this concept could include reporter genes that are detectable by other means, such as a simple visual color-change system.


Figure 2: Coupling the induced promoters of P. aeruginosa with reporting constructs (inserts 1 and 2)
fig1


Design Considerations


In our system, the lasR and rhlR genes are expressed from a high efficiency constitutive promoter and ribosome binding site (RBS). Given this high steady-state expression of LasR and RhlR proteins, we predict that the autoinducers PAI-1 and PAI-2 will be rate-limiting for the induction of the reporter genes. Therefore, reporter gene expression should commence rapidly upon the binding of PAI-1 or PAI-2 to their respective R-protein.



[1] Latifi A, Winson MK, Foglino M, Bycroft Bw, Stewart GS, Lazdunski A, et al. Multiple homologues of LuxR and LuxI control expression of virulence determinants and secondary metabolites through quorum sensing in Pseudomonas aeruginosa PAO1. Mol Microbiol 1995;17:333-43.

[2] Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, et al. Complete genome sequence ofPseudomonas aeruginosa PA01, an opportunistic pathogen. Nature. 2000;406:959–964.

[3] Wagner VE, Bushnell D, Passador L, Brooks AI, Iglewski BH. Microarray analysis ofPseudomonas aeruginosa quorum-sensing regulons: Effects of growth phase and environment. J Bacteriol. 2003;185:2080–2095.

[4] Pesci EC, Pearson JP, Seed PC, Iglewski BH. Regulation of las and rhl quorum sensing inPseudomonas aeruginosa. J Bacteriol 1997;179:3127-32.



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