Team:Toulouse/Project/binding

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                <li style="padding-right:0px"><a href="http://2014.igem.org/Team:Toulouse/Team" class="navlink" style="color:white;">ACKNOWLEGDEMENT<i style="padding-left: 10px;font-size: 14px;font-family: verdana;">></i> </a>
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                                                <li class="lisimpledrop"><a href="http://2014.igem.org/Team:Toulouse/acknowlegdement/attribution">Attribution</a></li>
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                        <li class="lisimpledrop"><a href="http://2014.igem.org/Team:Toulouse/acknowlegdement/sponsor">Sponsor</a></li>
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       <h2>Binding</h2>
       <h2>Binding</h2>
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       <p>Proin sollicitudin nibh ut dapibus vulputate.</p>
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       <p>To be attached to the fungal cell wall</p>
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<center><img style="width:700px; " src="http://2014.igem.org/wiki/images/e/e1/Bindingresume.png"></center>
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<p class="legend">Figure 1: Schema of the binding module</p>
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         <p class="texte"> The second step in the SubtiTree optimization matches with the <B> binding ability </B> of our bacterium. Thus, we design a chimeric protein (BBa_K1364005) able to make <B> a bridge between bacterial peptidoglycan and fungal chitin </B>, the main component of the pathogen’s cell wall. According to the Imperial College of London 2010 iGEM team, we use CWB domain of LytC protein to bind our chimeric protein to <I> Bacillus subtilis </I> cell wall. On the other side of our protein, we add the fragment of GbpA from <I> Vibrio Cholerae </I>, which is known to recognize N-Acetyl Glucosamine oligosaccharides called chitin.
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         <p class="texte"> In order to be highly efficient in the fight against the pythopathogen <i>Ceratocystis platani</i>, our optimized bacterium has to be anchored to the fungus.  
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Thus, we designed a chimeric protein (<a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>) capable of building
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a <B>bridge between the bacterial peptidoglycan and the fungal chitin</B>, the main component of the pathogen’s cell wall.  
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According to the work of <a href="http://2010.igem.org/Team:Imperial_College_London"target="_blank">the Imperial College 2010</a> iGEM team,  
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we used the Cell Wall Binding (CWB) domain of the <a href="http://www.uniprot.org/uniprot/Q02114"_blanck">LytC</a> protein (coding for a N-acetylmuramoyl-L-alanine amidase) to attach our chimeric protein  
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to the <I>Bacillus subtilis</I> cell wall. On the other side of our protein, we added the domain 4 of <a href="http://www.uniprot.org/uniprot/Q9KLD5"_blanck">GbpA</a> from <I>Vibrio cholerae</I>, which is known to  
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recognize chitin.
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<br>
<br>
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<B class="title2"> More information about this module </B>
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<p class="title1"> More information about this module </p>
<p class="texte">  
<p class="texte">  
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<br>The Binding Module ORF is composed of 3 sections:
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The open reading frame of the Binding Module is composed of 3 sections:</p>
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<B> <br>- Anchor section </B>: the CWB (Cell Wall Binding) is a LytC domain put on 5' of our chimeric protein gene. As previously used by the Imperial College of London 2010 iGEM team, we retain the first 318 bp. We can note the presence of the signal peptide at the beginning from 1 to 24 bp.  
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<B> <br>- Chitin Binding Domain (CBD) section </B:  the Domain 4 of GbpA from Vibrio Cholerae is able to bind to N-Acetyl Glucosamine oligosacchararides. Also, the last base pairs in 3' of our gene is composed by a part of the GbpA sequence (from 423 to 484 bp).
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<ul>
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<B> <br>- Helical Linker </B>: according to the work of the Imperial College of London 2010 iGEM team, we use the same six amino acids sequence (SRGSRA) to make a bridge between the Anchor section and the Chitin Binding section.
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<li class="tree"><p class="texte"><B>Anchor section</B>: the CWB (Cell Wall Binding) domain is extracted from LytC gene and composes the 5' side of our binding module. As previously described by the Imperial College of London 2010 iGEM team, we kept the first 318 bp. We can note the presence of the signal peptide at the beginning of the sequence, from 1 to 24 bp.</p></li>
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<li class="tree"><p class="texte"><B>Chitin Binding Domain (CBD) section</b>:  the domain 4 of GbpA from <I>V. cholerae </I> is able to bind to N-Acetyl Glucosamine oligosacchararides. Also, the 3' side of our gene is composed by a part of the GbpA sequence (from 423 to 484 bp).</p></li>
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<li class="tree"><p class="texte"><B>Helical Linker</B>: According to the work of the 2010 Imperial College of London iGEM team, we used the same six amino acids sequence (SRGSRA) to make a bridge between the Anchor section and the Chitin Binding section.
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</p></li></ul>
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<center style="margin:-30px;"><img style="width:500px; " src="http://2014.igem.org/wiki/images/a/a0/Binding.png"></center>
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<p class="legend">Figure 2: Binding gene composition</p>
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<br></br>
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<p class="texte">
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The sequence has been designed <i>in silico</i> and codon optimized for the transcription in <i>B. subtilis</i>.  
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<B class="title1">Final construction</B>
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<B class="title2">Final construction: (see also the section “Parts” of the wiki to have more details about the intermediate parts)</B>
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<B class="title2">(More details about the intermediate parts <a href="http://2014.igem.org/Team:Toulouse/Result/parts#select2"target="_blank">Here</a>)</B>  
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<p class="texte">
<p class="texte">
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<br>To introduce the Binding Module in Bacillus subtilis chromosome, we insert the ORF Binding Module in pSBBS4S plasmid (BBa_K823022), from the LMU-Munich 2012 iGEM team, with:
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<br>The binding module has been placed under the control of P<sub>veg</sub> (<a href="http://parts.igem.org/Part:BBa_K143012"target="_blank">BBa_K143012</a>),
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<br>- <B> Regulatory section </B>: after the ORF Binding Module, we include Pveg (BBa_K143012) as strong promoter and iGEM RBS consensus (BBa_K090505).
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a strong constitutive promoter and we used a consensus RBS (<a href="http://parts.igem.org/Part:BBa_K090505"target="_blank">BBa_K090505</a>) as well as a
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<br>- <B> Transcription end </B> : we use a double terminator (BBa_B0015)
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double terminator (<a href="http://parts.igem.org/Part:BBa_B0015"target="_blank">B0015</a>).
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<br> SCHEMA A RAJOUTER
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<center style="margin:20px;"><img style="width:500px; " src="http://2014.igem.org/wiki/images/f/f5/BBa_K1364005.png"></center>
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<p class="legend">Figure 3: Binding gene construction</p>
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<center><a href="http://2014.igem.org/Team:Toulouse/Result/experimental-results"> <img src="http://parts.igem.org/wiki/images/f/fe/Jump.jpg"> </a></center>
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<p class="title1">References</p>
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<ul>
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<li class="tree"><p class="texte">M. Desvaux, E. Dumas, I. Chafsey, and M. Hébraud.<b> Protein cell surface display in Gram-positive bacteria: from single protein to macromolecular protein structure </b>. FEMS Microbiol. Lett. 256, 1–15 (2006). </p></li>
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<li class="tree"><p class="texte">E. Wong, G. Vaaje-Kolstad, A. Ghosh, R. Hurtado-Guerrero, PV. Konarev, AF. Ibrahim, DI. Svergun, VG. Eijsink, NS. Chatterjee, and DM. van Aalten. <b>The <i>Vibrio cholerae</i> colonization factor GbpA possesses a modular structure that governs binding to different host surfaces</b>. PLoS Pathog. 8, e1002373 (2012).</p></li>
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<li class="tree"><p class="texte">H. Yamamoto, S. Kurosawa, and J. Sekiguchi. <b>Localization of the vegetative cell wall hydrolases LytC, LytE, and LytF on the <i>Bacillus subtilis</i> cell surface and stability of these enzymes to cell wall-bound or extracellular proteases</b>.  J. Bacteriol. 185, 6666–6677 (2003).</p></li>
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     <a href="http://2014.igem.org/Team:Toulouse/Project/Overviews" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Overviews
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     <a href="http://2014.igem.org/Team:Toulouse/Project/Chemotaxis" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Chemotaxis
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     <a href="http://2014.igem.org/Team:Toulouse/Project/Fungicides" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;
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color:#666; font-size:18px;">Chemotaxis</br>
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color:#666; font-size:18px;">Fungicides</br>
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Latest revision as of 03:00, 18 October 2014