Team:Freiburg bioware/Project

From 2009.igem.org

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

Revision as of 08:20, 21 October 2009

FREiGEM



We focused our project on coupling and optimizing the characteristics of a restriction endonuclease with short oligonucleotides to develop a programmable and highly specific enzyme-oligo-complex. As a restriction endonuclease we chose the cleavage domain of the well studied endonuclease FokI from Flavobacterium okeanokoites. Normally FokI acts as a homodimer, each dimer divided in cleavage and restriction domain. Chandrasegaran and Miller have already made experiments to uncouple the cleavage and restriction domains of FokI and created a novel site-specific endonuclease by linking the cleavage domain to zinc finger proteins.
For our project we generated two Fok heterodimers (Miller, Nature biotech, 2007). For the catalytic active Fok partner, named Fok_a, the first 1158 nucleotides, i.e. the recognition domain, were deleted and glutamate 490 was switched to lysine (GAA->AAA) as well as isoleucine 538 to lysine (ATC->AAA) for the heterodimer formation. For the catalytic inactive Fok partner, named Fok_i, the heterodimeric amino acids glutamine 486 was switched to glutamate (CAA->GAA) and isoleucine 499 to leucine (ATC->CTG) and the catalytic amino acids aspartate 450 was switched to alanine (GAC->GCG) and aspartate 467 to alanine (GAT->GCG).

Association of linker FluA and Dig with DNA and Fok_a and Fok_i monomers

The two heterodimeric partners were fused to different anticalins binding different adapter molecules. Thus Fok_i is fused to anticalin on Fluorescein and Fok_a to anticalin on Digoxigenin. These adapter molecules are linked to oligonucleotides mediating the binding of the DNA site of interest. Now the heterodimerization comes into play. If the different Fok_i and Fok_a constructs bind their target oligos and come together, the inactive domain will serve simply as an activator of the active domain, cutting only one strand of the DNA. In our 3D models we showed that Fok domains are positioned in such a way that Fok_a will cut the DNA and Fok_i the modified oligonucleotide. Thus the inactivation of Fok_i allows the reuse of our oligonucleotides. Different linkers were designed and fused between cleavage domain and binding protein to test the optimal distance to preserve the most possible flexibility and most possible precision of the heterodimeric Foks.





Modelling

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3D-Modelling

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Fok mit Aktivität

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Fok Monomer

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Purification

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In vivo Expression

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AGO

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FOS

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Cloning strategy

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Expression

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