Team:HIT-Harbin/project/part1

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HIT-Harbin

Part1: Detecting

The detecting system is constructed to detect the existence of Staphylococcus aureus，which is based on the global regulator of virulence, agr quorum sensing system of S.aureus that modulates the expression of virulence factors in response to autoinducing peptides (AIPs)[1]. The detecting system we constructed is mainly composed of agrA and agrC.

In the pathogenic species Staphylococcus aureus, the extracellular signal of the quorum sensing system is a thiolactone-containing cyclic peptides pheromone (AIP), whose sequence varies among the different staphylococcus strains. The polymorphism in the amino acid sequence of the AIP and of its corresponding receptor (AgrC) divides S.aureus strains into four major groups. The AIPs belonging to different groups are usually mutually inhibitory[

AgrC is a transmembrane protein, which is the sensor molecule of a typical two-component signal system in S.aureus. AgrC possesses several key amino acid motifs typical of histidine protein kinase sensor. The AgrC sensor kinase can specifically binds to corresponding AIP, which secreted only from specific S.aureus, and the composite of AgrC and AIP then leads to phosphorylation of AgrA. AgrA in its phosphorylated sate activates transcription from both P2 and P3, leading to the production of GFP and 3OC6HSL. Thus we can detect the presence of S.aureus expediently by observing the expression of GFP. The figure shows the mechanism of our detecting system in E.coli.

There is a trouble that the agr system belongs to S.aureus, but we hope this system works in E.coli, but . Therfore, we analyze the topology structure of AgrC and AgrA. Staphylococcus aureus AgrA, the transcriptional component of a quorum sensing system and global regulator of virulence that up-regulates secreted virulence factors and down-regulates cell wall-associated proteins, can bind in both the P2 and P3 promoter regions of the agr locus. The structure of AgrA, described by an online software PDB (Protein Data Bank), has ten β strands arranged into three antiparallel β sheets and a small α helix. The sheets are arranged roughly parallel to each other in an elongated β-β-β sandwich. A hydrophobic five-stranded β sheet (sheet 2: β3-β7) is at the center of the domain with two smaller amphipathic β sheets (sheet 1: β1-β2 and sheet 3: β8-β10) positioned on either side.

Fig 2. Structure of the Staphylococcus aureus AgrA bounding to DNA

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-<p>&nbsp;&nbsp;&nbsp;&nbsp;Staphylococcus aureus infections are major causes of morbidity and mortality in community and hospital settings. Consequently, the emergence of methicillin-resistant and, more recently, vancomysin-resistant strains of S.aureus represents an enormous threat to public health. Since bacterial sensors are attracting more and more biologists' attention owing to its' specific, fast and accurate detecting, we plan to construct a E.coli biofilm consisting of two different engineered populations(namely biosensor and killer), which are designed to detect and eradicate S.aureus, respectively. The two engineered populations communicate with each other by AHL signal transduction. We hope that compartmentalization of functions can lessen metabolism load and cross-reactions interfere, and achieve the assembly of different functions in bacterial level. The whole system comprises sensing, killing and biofilm formation devices. </p>
+<p>&nbsp;&nbsp;&nbsp;&nbsp;The detecting system is constructed to detect the existence of Staphylococcus aureus，which is based on the global regulator of virulence, agr quorum sensing system of S.aureus that modulates the expression of virulence factors in response to autoinducing peptides (AIPs)[1]. The detecting system we constructed is mainly composed of agrA and agrC. </p>
-<p>&nbsp;&nbsp;&nbsp;&nbsp;Detecting device: to detect the existence of S.aureus through sensing the AIPs secreted only from S.aureus, and the report will be shown by the expression of GFP.</p>
+<p>&nbsp;&nbsp;&nbsp;&nbsp;In the pathogenic species Staphylococcus aureus, the extracellular signal of the quorum sensing system is a thiolactone-containing cyclic peptides pheromone (AIP), whose sequence varies among the different staphylococcus strains. The polymorphism in the amino acid sequence of the AIP and of its corresponding receptor (AgrC) divides S.aureus strains into four major groups. The AIPs belonging to different groups are usually mutually inhibitory[</p>
-<p>&nbsp;&nbsp;&nbsp;&nbsp;Killing device: to eradicate S.aureus through the production and release of lysostaphin.</p>
+<p>&nbsp;&nbsp;&nbsp;&nbsp;AgrC is a transmembrane protein, which is the sensor molecule of a typical two-component signal system in S.aureus. AgrC possesses several key amino acid motifs typical of histidine protein kinase sensor. The AgrC sensor kinase can specifically binds to corresponding AIP, which secreted only from specific S.aureus, and the composite of AgrC and AIP then leads to phosphorylation of AgrA. AgrA in its phosphorylated sate activates transcription from both P2 and P3, leading to the production of GFP and 3OC6HSL. Thus we can detect the presence of S.aureus expediently by observing the expression of GFP. The figure shows the mechanism of our detecting system in E.coli.</p>
-<p>&nbsp;&nbsp;&nbsp;&nbsp;Biofilm formation device: to enhance biofilm formation by over-expression of yddV, a di-guanylate cyclase, which catalyzes GTP into c-di-GMP.</p>
+<p>&nbsp;&nbsp;&nbsp;&nbsp;There is a trouble that the agr system belongs to S.aureus, but we hope this system works in E.coli, but . Therfore, we analyze the topology structure of AgrC and AgrA. Staphylococcus aureus AgrA, the transcriptional component of a quorum sensing system and global regulator of virulence that up-regulates secreted virulence factors and down-regulates cell wall-associated proteins, can bind in both the P2 and P3 promoter regions of the agr locus. The structure of AgrA, described by an online software PDB (Protein Data Bank), has ten β strands arranged into three antiparallel β sheets and a small α helix. The sheets are arranged roughly parallel to each other in an elongated β-β-β sandwich. A hydrophobic five-stranded β sheet (sheet 2: β3-β7) is at the center of the domain with two smaller amphipathic β sheets (sheet 1: β1-β2 and sheet 3: β8-β10) positioned on either side.</p>
 <img  src="https://static.igem.org/mediawiki/2012/0/09/Op.jpg">
-<font size="2"><p>Fig. 1 Schematic of “S.aureus monitor” system. The AgrA protein is phosphorylated when AIP binds to AgrC protein across the membrane. P2(P3) is activated by phosphorylated AgrA, leading the production and release of 3OC6HSL. PluxI promoter is induced by LuxR-3OC6HSL complex, causing the production of lysostaphin and starting the time-delay device(PKU iGEM 2010). After a certain time, lysis E7 protein(NTU iGEM 2009) is produced. After the E7 protein attains the threshold concentration that causes the chassis to lyse, the accumulated lysostaphin is released into the exogenous environment and kills S.aureus.
+<font size="2"><p>Fig 2. Structure of the Staphylococcus aureus AgrA bounding to DNA<p><font>
-T5:constitutive promoter; P2(P3):AgrA~P inducible promoter; Ptrc:IPTG inducible promoter; PluxI:LuxR-3OC6HSL complex inducible promoter; AIP: produced by Staphylococcus aureus<p><font>
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