Team:Freiburg bioware/Project

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<div style="text-align: left;">We focused our project on coupling and optimizing the characteristics
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of a restriction endonuclease with short oligonucleotides to develop a
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programmable and highly specific enzyme-oligo-complex. As a restriction
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endonuclease we chose the cleavage domain of the well studied
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endonuclease FokI from Flavobacterium okeanokoites. Normally FokI acts
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as a homodimer, each dimer divided in cleavage and restriction domain.
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Chandrasegaran and Miller have already made experiments to uncouple the
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cleavage and restriction domains of FokI and created a novel
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site-specific endonuclease by linking the cleavage domain to zinc
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finger proteins.<br>
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For our project we generated two Fok heterodimers (Miller, Nature
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biotech, 2007). For the catalytic active Fok partner, named Fok_a, the
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first 1158 nucleotides, i.e. the recognition domain, were deleted and
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glutamate 490 was switched to lysine (GAA-&gt;AAA) as well as
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isoleucine 538 to lysine (ATC-&gt;AAA) for the heterodimer
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formation.
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For the catalytic inactive Fok partner, named Fok_i, the heterodimeric
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amino acids glutamine 486 was switched to glutamate (CAA-&gt;GAA)
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and
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isoleucine 499 to leucine (ATC-&gt;CTG) and the catalytic amino
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acids
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aspartate 450 was switched to alanine (GAC-&gt;GCG) and aspartate
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467
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to alanine (GAT-&gt;GCG).<br>
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<br>
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<table
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style="text-align: left; width: 509px; height: 378px; float: left;"
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border="0" cellpadding="0" cellspacing="0">
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  <tbody>
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    <tr>
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      <td><img style="width: 503px; height: 338px;"
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alt=""
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src="https://static.igem.org/mediawiki/2009/0/04/Freiburg09_Foka_foki_in_action.JPG"></td>
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    </tr>
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    <tr>
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      <td style="background-color: rgb(50, 122, 153);">Association
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of linker FluA and Dig with DNA and Fok_a and Fok_i monomers</td>
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    </tr>
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  </tbody>
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</table>
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<br>
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The two heterodimeric partners were fused to different anticalins
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binding different adapter molecules. Thus Fok_i is fused to anticalin
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on Fluorescein and Fok_a to anticalin on Digoxigenin. These adapter
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molecules are linked to oligonucleotides mediating the binding of the
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DNA site of interest. Now the heterodimerization comes into play. If
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the different Fok_i and Fok_a constructs bind their target oligos and
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come together, the inactive domain will serve simply as an activator of
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the active domain, cutting only one strand of the DNA. In our 3D models
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we showed that Fok domains are positioned in such a way that Fok_a will
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cut the DNA and Fok_i the modified oligonucleotide. Thus the
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inactivation of Fok_i allows the reuse of our oligonucleotides.
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Different linkers were designed and fused between cleavage domain and
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binding protein to test the optimal distance to preserve the most
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possible flexibility and most possible precision of the heterodimeric
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Foks.
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Revision as of 13:27, 21 October 2009

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