Team:HIT-Harbin/project/part3

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<li class="page_item page-item-39 "><a href="https://2012.igem.org/Team:HIT-Harbin/team" title="Alternate Home Page">TEAM</a>
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/team" title="OVERVIEW">OVERVIEW</a>
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/team" title="OVERVIEW">OVERVIEW</a></li>
<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/team/undergraduates" title="UNDERGRADUATES">UNDERGRADUATES</a></li>
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/team/abouttheuniversity" title="ABOUT THE UNIVERSITY">ABOUT THE UNIVERSITY</a>
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/team/abouttheuniversity" title="ABOUT THE UNIVERSITY">ABOUT THE UNIVERSITY</a></li>
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project" title="OVERVIEW">OVERVIEW</a></li>
<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project" title="OVERVIEW">OVERVIEW</a></li>
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project/part1" title="PART 1">PART 1</a></li>
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project/part1" title="BIOSENSOR">BIOSENSOR</a></li>
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project/part2" title="PART 2">PART 2</a></li>
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project/part2" title="BIOKILLER">BIOKILLER</a></li>
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project/part3" title="PART 3">PART 3</a></li>
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project/part3" title="BIOFILM">BIOFILM</a></li>
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project/model" title="PART 3">MODEL</a></li>
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project/model" title="MODELING">MODELING</a></li>
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project/application"  
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project/application" title="APPLICATION">APPLICATION</a></li>
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<li class="page_item page-item-39 c"><a href="PARTS.html" title="PARTS<">PARTS</a>
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<li class="page_item page-item-39 "><a href="https://2012.igem.org/Team:HIT-Harbin/parts" title="PARTS">PARTS</a></li>
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<li class="page_item page-item-39 "><a href="https://2012.igem.org/Team:HIT-Harbin/team/safety" title="SAFETY">SAFETY </a>
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<li class="page_item page-item-39 "><a href="https://2012.igem.org/Team:HIT-Harbin/team/safety" title="SAFETY">SAFETY </a></li>
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<li class="page_item page-item-39 "><a href="https://2012.igem.org/Team:HIT-Harbin/project/notebook/diary" title="Alternate Home Page">NOTEBOOK</a>
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project/notebook/diary" title="DIARY">DIARY</a></li>
<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project/notebook/diary" title="DIARY">DIARY</a></li>
<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/notebook/protocol" title="PROTOCOL">PROTOCOL</a></li>
<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/notebook/protocol" title="PROTOCOL">PROTOCOL</a></li>
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/notebook/attribution" title="Attributions<">Attributions</a></li>
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/notebook/attribution" title="ATTRIBUTION">ATTRIBUTION</a></li>
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<li class="page_item page-item-39 "><a href="https://2012.igem.org/Team:HIT-Harbin/humanpractice/lecture" title="HUMAN PRACTICE">HUMAN PRACTICE</a>
<li class="page_item page-item-39 "><a href="https://2012.igem.org/Team:HIT-Harbin/humanpractice/lecture" title="HUMAN PRACTICE">HUMAN PRACTICE</a>
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/humanpractice/lecture" title="LECTURE">LECTURE</a>
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/humanpractice/lecture" title="LECTURE">LECTURE</a></li>
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/humanpractice/software" title="THE SOFTRWARE">THE SOFTRWARE</a></li>
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/humanpractice/software" title="SOFTRWARE">SOFTRWARE</a></li>
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<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/humanpractice/song" title="THE SONG">THE SONG</a></li>
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Part3: Biofilm</div>  
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Device3: Biofilm</div>  
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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;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. In our system, detecting part and killing part are separated into two different engineered <em>E.coli</em> populations. As we know, people in a small room communicate more conveniently than those in a big one. We assumed that signal transduction between <em>E.coli</em> 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;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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<img  src="https://static.igem.org/mediawiki/2012/2/2b/B1.png">
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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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<font size="2"><p>Fig.1 the biobrick of biofilm formation device</p></font>
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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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<img  src="https://static.igem.org/mediawiki/2012/0/09/Op.jpg">
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<p>&nbsp;&nbsp;&nbsp;&nbsp;We introduce a gene <em>yddV</em>, which is under the regulation of Ptrc promoter (IPTG-inducible) (Fig. 1). <em>yddV</em> is a Diguanylate Cyclase-Genomic. The product of gene <em>yddV</em> has diguanylate cyclase (DGC) activity. DGC uses 2 GTP to form a Bisbis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). </p>
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<font size="2"><p>Fig 2. Structure of the Staphylococcus aureus AgrA bounding to DNA<p><font>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;C-di-GMP is a global second messenger in bacteria. Biofilm formation of <em>E.coli</em> 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 concentrations 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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<img  src="https://static.igem.org/mediawiki/2012/d/d1/B2.png">
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<p>&nbsp;&nbsp;&nbsp;&nbsp;In order to check whether the construct is ligated to the plasmid, the biobrick region (1604bp) is PCR-amplified using primers B1(5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGAG-3') and B2(5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTA-3'). As shown in Fig. 2, the amount of the amplified sequence is almost same as expected. <em>yddV</em> is maximal when an exponentially growing culture (A600=0.3) was treated with 1mM IPTG for 2h. As shown in Fig. 3, an increase in the expression of <em>yddV</em> was evident when 1mM IPTG was added to the medium.</p>
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<img  src="https://static.igem.org/mediawiki/2012/a/a2/B3.png">
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<p>&nbsp;&nbsp;&nbsp;&nbsp;Biofilm assays were carried out at 37℃. Cultures were grown in LB to exponential phase(A600=0.3). The culture of the <em>E.coli</em> cells carrying the plasmid pYddV was split in two test-tubes, each containing 5ml medium. 1mM IPTG was added to one culture, and the other was left untreated. At the same time, 5ml culture of the <em>E.coli</em> cells not carrying the pYddV was transferred into a test-tube. After 24h of treatment, visualization of attached cells was performed by removing the cell culture, staining the well with 1.0% crystal violet for 20min, and rinsing the well three times with distilled water. It was evident that a significant increase in biofilm formation in LB cultures of the <em>E.coli</em> cells carrying the pYddV in comparison with those <em>E.coli</em> cells not carrying the pYddV(Fig. 4).</p>
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<img  src="https://static.igem.org/mediawiki/2012/f/ff/B4.png">
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<p>&nbsp;&nbsp;&nbsp;&nbsp;Next, we will determine the composition of two engineered bacteria in the biofilm we constructed. Moreover, we will further test whether the biofilm strengthen the intra- and inter-species signal transduction.</p>
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<br><br><br><br>
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<div class="post-title">
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<a>Reference</a>
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</div>
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<div class="post-excerpt">
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<p>[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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</div>  
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<p><a id="backtotop" href="https://2012.igem.org/Team:HIT-Harbin/project/part3#header">Back to Top</a><p></div></div>
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Latest revision as of 05:29, 23 October 2012

HIT-Harbin

Device3: 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. 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 those in a big one. We assumed 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 Bisbis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP).

    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 concentrations of c-di-GMP promotes bacteria to form more cellulose and fimbriae, which enhances the biofilm formation and decrease the motility of bacteria.

    In order to check whether the construct is ligated to the plasmid, the biobrick region (1604bp) is PCR-amplified using primers B1(5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGAG-3') and B2(5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTA-3'). As shown in Fig. 2, the amount of the amplified sequence is almost same as expected. yddV is maximal when an exponentially growing culture (A600=0.3) was treated with 1mM IPTG for 2h. As shown in Fig. 3, an increase in the expression of yddV was evident when 1mM IPTG was added to the medium.

    Biofilm assays were carried out at 37℃. Cultures were grown in LB to exponential phase(A600=0.3). The culture of the E.coli cells carrying the plasmid pYddV was split in two test-tubes, each containing 5ml medium. 1mM IPTG was added to one culture, and the other was left untreated. At the same time, 5ml culture of the E.coli cells not carrying the pYddV was transferred into a test-tube. After 24h of treatment, visualization of attached cells was performed by removing the cell culture, staining the well with 1.0% crystal violet for 20min, and rinsing the well three times with distilled water. It was evident that a significant increase in biofilm formation in LB cultures of the E.coli cells carrying the pYddV in comparison with those E.coli cells not carrying the pYddV(Fig. 4).

    Next, we will determine the composition of two engineered bacteria in the biofilm we constructed. Moreover, we will further test whether the biofilm strengthen the intra- and inter-species signal transduction.





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.

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