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| - | <h1><font color="green"> A race between two different strategies was used to obtain the first part </font></h1> <HR>
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| - | <p style="line-height:1.5em">
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| - | In E. coli the curli-producing system is organized in two divergeant operons with the genes
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| - | csgA, csgB and csgC on one side and csgD, csgE, csgF and csgG of the other one. So to
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| - | achieve the first approach of the project, it is necessary to begin by the creation of three "sub-
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| - | parts": the first one would be the part with the sole promoter PrcnA, the second would be the
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| - | part with the genes csgA and csgB and final one would be the part with the genes csgE, csgF
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| - | and csgG.
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| - | <br/> <br/>
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| - | </p>
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| - | <p style="line-height:1.5em">
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| - | The synthesis of this first DNA part (the cobalt-inducible promoter Prcn-csgBAEFG, which is
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| - | designed to induce formation of a biofilm and to promote cell adhesion) was conceived as as a
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| - | competition between two methods: a completely synthetic approach, and a "manual" method
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| - | involving PCR steps followed by ligations. <br/>
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| - | </p>
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| - | <ul style="list-style-type:circle;margin-left:30%;">
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| - | <li style="line-height : 1.5em"> The first approach consist in ordering the whole part at a private company (Genecust).</li>
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| - | <br/>
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| - | <li style="line-height : 1.5em"> The second approach was to made it directly at the bench, this approach included three
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| - | steps: first the amplification by PCR of each of the sub parts, second a mutagenesis
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| - | step to remove all the natural internal EcoRI sites located in the sub parts, and finally
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| - | the ligation of these parts.</li>
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| - | <br/>
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| - | </ul>
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| - | <p style="line-height:1.5em">
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| - | Both approches were initiated at the same time, and if the second one allowed us to obtain
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| - | PCR amplifications of the correct size (see figure) and a complete Prcn-csgBAEFG
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| - | construction, unfortunately sequencing analysis revealed unexpected mutations that were not
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| - | removed before reception of the whole part made by Genecust. In brief the "click and order"
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| - | strategy wins the race !
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| - | </p>
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| - | <img src="https://static.igem.org/mediawiki/2011/1/19/Achiev2.jpg"/>
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| - | <p style="line-height:1.5em; float : left; margin-left : 58%; margin-top : -100px; width : 350px">
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| - | PCR amplification of sub-parts : csgEFG 1.8kb (lane 1), csgBA 1kb (lane 3) et Prcn 0.5kb
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| - | (lane 5) and PCR negative controls (lanes 2, 4 and 6)
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| - | </p>
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| - | </div>
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