Team:HIT-Harbin/project/part3

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<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>
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<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>
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Biofilms are groups of cells at an interface cemented together by polysaccharides, protein, DNA and lipids. It enhances bacteria’s resistance to environmental stress and is able to perform more complex transformations[1]. In our system, detecting part and killing part are separated into two different engineered E.coli populations. As we know, people in a small room communicate more conveniently than in a big one. We assume that signal transduction between E.coli is similar to people’s communication. In order to strengthen signal transduction between two communities, we construct an enhanced consortium biofilm. </p>
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<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>
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<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>
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              <p><br> Fig. 1 the biobrick of biofilm formation device.</p>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;We introduce a gene yddV, which is under the regulation of Ptrc promoter (IPTG-inducible) (Fig. 1). yddV is a Diguanylate Cyclase-Genomic. The product of gene yddV has diguanylate cyclase (DGC) activity. DGC uses 2 GTP to form a bis-(3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP)[2]. </p>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;C-di-GMP is a global second messenger in bacteria. Biofilm formation of E.coli is manipulable by varying c-di-GMP concentrations. When the c-di-GMP level stays low, there is little biofilm and the bacteria is dispersive. High concentration of c-di-GMP promotes bacteria to form more cellulose and fimbriae, which enhances the biofilm formation and decrease the motility of bacteria.</p>
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<p><br><br>Reference:
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[1] Hyun-Dong Shin, Shara McClendon, Trinh Vo, and Rachel R. Chen．Escherichia coli Binary Culture Engineered for Direct Fermentation of Hemicellulose to a Biofuel[J]. Applied and Environmental Microbiology, 2010, 76(24):8150-8159.</p>
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<p>[2] M. Marcela Me´ndez-Ortiz, Mamoru Hyodo, Yoshihiro Hayakawa, and Jorge Membrillo-Herna´ndez．Genome-wide Transcriptional Profile of Escherichia coli in Response to High Levels of the Second Messenger 3,5-Cyclic Diguanylic Acid[J]. The Journal of Biological Chemistry, 2006, 281(12):8090-8099.</p>
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<img  src="https://static.igem.org/mediawiki/2012/0/09/Op.jpg">
<img  src="https://static.igem.org/mediawiki/2012/0/09/Op.jpg">
<font size="2"><p>Fig 2. Structure of the Staphylococcus aureus AgrA bounding to DNA<p><font>
<font size="2"><p>Fig 2. Structure of the Staphylococcus aureus AgrA bounding to DNA<p><font>

Revision as of 06:38, 24 September 2012

HIT-Harbin

Part3: Biofilm

     Biofilms are groups of cells at an interface cemented together by polysaccharides, protein, DNA and lipids. It enhances bacteria’s resistance to environmental stress and is able to perform more complex transformations[1]. In our system, detecting part and killing part are separated into two different engineered E.coli populations. As we know, people in a small room communicate more conveniently than in a big one. We assume that signal transduction between E.coli is similar to people’s communication. In order to strengthen signal transduction between two communities, we construct an enhanced consortium biofilm.


Fig. 1 the biobrick of biofilm formation device.

    We introduce a gene yddV, which is under the regulation of Ptrc promoter (IPTG-inducible) (Fig. 1). yddV is a Diguanylate Cyclase-Genomic. The product of gene yddV has diguanylate cyclase (DGC) activity. DGC uses 2 GTP to form a bis-(3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP)[2].

    C-di-GMP is a global second messenger in bacteria. Biofilm formation of E.coli is manipulable by varying c-di-GMP concentrations. When the c-di-GMP level stays low, there is little biofilm and the bacteria is dispersive. High concentration of c-di-GMP promotes bacteria to form more cellulose and fimbriae, which enhances the biofilm formation and decrease the motility of bacteria.



Reference: [1] Hyun-Dong Shin, Shara McClendon, Trinh Vo, and Rachel R. Chen．Escherichia coli Binary Culture Engineered for Direct Fermentation of Hemicellulose to a Biofuel[J]. Applied and Environmental Microbiology, 2010, 76(24):8150-8159.

[2] M. Marcela Me´ndez-Ortiz, Mamoru Hyodo, Yoshihiro Hayakawa, and Jorge Membrillo-Herna´ndez．Genome-wide Transcriptional Profile of Escherichia coli in Response to High Levels of the Second Messenger 3,5-Cyclic Diguanylic Acid[J]. The Journal of Biological Chemistry, 2006, 281(12):8090-8099.

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

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