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Team:Toulouse/Project/binding
From 2014.igem.org
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<h2>Binding</h2> | <h2>Binding</h2> | ||
| - | <p>To be attached to the fungal wall</p> | + | <p>To be attached to the fungal cell wall</p> |
</div> | </div> | ||
</div> | </div> | ||
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<!--Short description : à changer!!!--> | <!--Short description : à changer!!!--> | ||
<center><img style="width:700px; " src="https://static.igem.org/mediawiki/2014/e/e1/Bindingresume.png"></center> | <center><img style="width:700px; " src="https://static.igem.org/mediawiki/2014/e/e1/Bindingresume.png"></center> | ||
| + | <p class="legend">Figure 1: Schema of the binding module</p> | ||
| - | <p class="texte"> In order to be highly efficient in the fight against the pythopathogen, our optimized bacterium has to be anchored to the fungus. Thus, we designed a chimeric protein (<a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>) capable of building a <B>bridge between the bacterial peptidoglycan and the fungal chitin</B>, the main component of the pathogen’s cell wall. According to the work of <a href="https://2010.igem.org/Team:Imperial_College_London"target="_blank">the Imperial College 2010</a> iGEM team, we used the Cell Wall Binding (CWB) domain of the LytC protein (coding for a N-acetylmuramoyl-L-alanine amidase) to attach our chimeric protein to the <I> | + | <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. |
| + | Thus, we designed a chimeric protein (<a href="http://parts.igem.org/Part:BBa_K1364005"target="_blank">BBa_K1364005</a>) capable of building | ||
| + | a <B>bridge between the bacterial peptidoglycan and the fungal chitin</B>, the main component of the pathogen’s cell wall. | ||
| + | According to the work of <a href="https://2010.igem.org/Team:Imperial_College_London"target="_blank">the Imperial College 2010</a> iGEM team, | ||
| + | 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 | ||
| + | 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 | ||
| + | recognize chitin. | ||
</p> | </p> | ||
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<p class="title1"> More information about this module </p> | <p class="title1"> More information about this module </p> | ||
<p class="texte"> | <p class="texte"> | ||
| - | The Binding Module | + | The open reading frame of the Binding Module is composed of 3 sections:</p> |
<ul> | <ul> | ||
<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> | <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> | ||
| - | <li class="tree"><p class="texte"><B>Chitin Binding Domain (CBD) section</b>: the domain 4 of GbpA from <I> | + | <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> |
<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. | <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. | ||
</p></li></ul> | </p></li></ul> | ||
<center style="margin:-30px;"><img style="width:500px; " src="https://static.igem.org/mediawiki/2014/a/a0/Binding.png"></center> | <center style="margin:-30px;"><img style="width:500px; " src="https://static.igem.org/mediawiki/2014/a/a0/Binding.png"></center> | ||
| + | <p class="legend">Figure 2: Binding gene composition</p> | ||
<br></br> | <br></br> | ||
<p class="texte"> | <p class="texte"> | ||
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<p class="texte"> | <p class="texte"> | ||
| - | <br>The binding module has been placed under the control of | + | <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>), |
| + | 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 | ||
| + | double terminator (<a href="http://parts.igem.org/Part:BBa_B0015"target="_blank">B0015</a>). | ||
| + | </p> | ||
<center style="margin:20px;"><img style="width:500px; " src="https://static.igem.org/mediawiki/2014/f/f5/BBa_K1364005.png"></center> | <center style="margin:20px;"><img style="width:500px; " src="https://static.igem.org/mediawiki/2014/f/f5/BBa_K1364005.png"></center> | ||
| + | <p class="legend">Figure 3: Binding gene construction</p> | ||
| + | |||
| + | <center><a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results"> <img src="https://static.igem.org/mediawiki/parts/f/fe/Jump.jpg"> </a></center> | ||
| + | |||
<p class="title1">References</p> | <p class="title1">References</p> | ||
<ul> | <ul> | ||
| - | <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> | + | <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> |
| - | <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 Vibrio cholerae colonization factor GbpA possesses a modular structure that governs binding to different host surfaces</b>. PLoS Pathog. 8, e1002373 (2012).</p></li> | + | <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> |
| - | <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 Bacillus subtilis cell surface and stability of these enzymes to cell wall-bound or extracellular proteases</b>. J. Bacteriol. 185, 6666–6677 (2003).</p></li> | + | <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> |
</ul> | </ul> | ||
Latest revision as of 03:00, 18 October 2014
Binding
To be attached to the fungal cell wall
Project > Binding
Figure 1: Schema of the binding module
In order to be highly efficient in the fight against the pythopathogen Ceratocystis platani, our optimized bacterium has to be anchored to the fungus. Thus, we designed a chimeric protein (BBa_K1364005) capable of building a bridge between the bacterial peptidoglycan and the fungal chitin, the main component of the pathogen’s cell wall. According to the work of the Imperial College 2010 iGEM team, we used the Cell Wall Binding (CWB) domain of the LytC protein (coding for a N-acetylmuramoyl-L-alanine amidase) to attach our chimeric protein to the Bacillus subtilis cell wall. On the other side of our protein, we added the domain 4 of GbpA from Vibrio cholerae, which is known to recognize chitin.
More information about this module
The open reading frame of the Binding Module is composed of 3 sections:
Anchor section: 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.
Chitin Binding Domain (CBD) section: the domain 4 of GbpA from V. cholerae 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).
Helical Linker: 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.
Figure 2: Binding gene composition
The sequence has been designed in silico and codon optimized for the transcription in B. subtilis.
Final construction (More details about the intermediate parts Here)
The binding module has been placed under the control of Pveg (BBa_K143012),
a strong constitutive promoter and we used a consensus RBS (BBa_K090505) as well as a
double terminator (B0015).
Figure 3: Binding gene construction
References
M. Desvaux, E. Dumas, I. Chafsey, and M. Hébraud. Protein cell surface display in Gram-positive bacteria: from single protein to macromolecular protein structure . FEMS Microbiol. Lett. 256, 1–15 (2006).
E. Wong, G. Vaaje-Kolstad, A. Ghosh, R. Hurtado-Guerrero, PV. Konarev, AF. Ibrahim, DI. Svergun, VG. Eijsink, NS. Chatterjee, and DM. van Aalten. The Vibrio cholerae colonization factor GbpA possesses a modular structure that governs binding to different host surfaces. PLoS Pathog. 8, e1002373 (2012).
H. Yamamoto, S. Kurosawa, and J. Sekiguchi. Localization of the vegetative cell wall hydrolases LytC, LytE, and LytF on the Bacillus subtilis cell surface and stability of these enzymes to cell wall-bound or extracellular proteases. J. Bacteriol. 185, 6666–6677 (2003).
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