Team:Freiburg bioware/Project

From 2009.igem.org

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</div>
</div>
<div class="art-PostContent">
<div class="art-PostContent">
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<div style="text-align: left;">We focused our project on coupling and optimizing the characteristics
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<div style="text-align: center;"></div>
 +
We focused our project on coupling and optimizing the characteristics
of a restriction endonuclease with short oligonucleotides to develop a
of a restriction endonuclease with short oligonucleotides to develop a
programmable and highly specific enzyme-oligo-complex. As a restriction
programmable and highly specific enzyme-oligo-complex. As a restriction
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cleavage and restriction domains of FokI and created a novel
cleavage and restriction domains of FokI and created a novel
site-specific endonuclease by linking the cleavage domain to zinc
site-specific endonuclease by linking the cleavage domain to zinc
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finger proteins.<br>
+
finger proteins.<br />
For our project we generated two Fok heterodimers (Miller, Nature
For our project we generated two Fok heterodimers (Miller, Nature
biotech, 2007). For the catalytic active Fok partner, named Fok_a, the
biotech, 2007). For the catalytic active Fok partner, named Fok_a, the
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aspartate 450 was switched to alanine (GAC-&gt;GCG) and aspartate
aspartate 450 was switched to alanine (GAC-&gt;GCG) and aspartate
467
467
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to alanine (GAT-&gt;GCG).<br>
+
to alanine (GAT-&gt;GCG).<br />
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<br>
+
<br />
 +
<div style="text-align: justify;"></div>
<table
<table
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  style="text-align: left; width: 509px; height: 378px; float: left;"
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  style="float: left; text-align: left; margin-top: 2px; margin-right: 20px; margin-bottom: 15px; background-color: rgb(205, 219, 229); width: 263px; height: 237px;"
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  border="0" cellpadding="0" cellspacing="0">
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  border="1" cellpadding="2" cellspacing="0">
   <tbody>
   <tbody>
     <tr>
     <tr>
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       <td><img style="width: 503px; height: 338px;"
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       <td><img style="width: 300px; height: 202px;"
  alt=""
  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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  src="https://static.igem.org/mediawiki/2009/0/04/Freiburg09_Foka_foki_in_action.JPG" /></td>
     </tr>
     </tr>
     <tr>
     <tr>
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       <td style="background-color: rgb(50, 122, 153);">Association
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       <td><small>Association of linker FluA and Dig with
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of linker FluA and Dig with DNA and Fok_a and Fok_i monomers</td>
+
DNA and</small> <small>Fok_a
 +
and Fok_i monomers</small></td>
     </tr>
     </tr>
   </tbody>
   </tbody>
</table>
</table>
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<br>
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<div style="text-align: justify;">The two heterodimeric
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The two heterodimeric partners were fused to different anticalins
+
partners were fused to different anticalins
binding different adapter molecules. Thus Fok_i is fused to anticalin
binding different adapter molecules. Thus Fok_i is fused to anticalin
on Fluorescein and Fok_a to anticalin on Digoxigenin. These adapter
on Fluorescein and Fok_a to anticalin on Digoxigenin. These adapter
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binding protein to test the optimal distance to preserve the most
binding protein to test the optimal distance to preserve the most
possible flexibility and most possible precision of the heterodimeric
possible flexibility and most possible precision of the heterodimeric
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Foks.
+
Foks.<br />
 +
<br />
 +
<br />
 +
<br />
 +
<br />
 +
<br />
 +
<br />
 +
<br />
 +
<table
 +
style="margin-top: 4px; margin-left: 20px; text-align: left; float: right; background-color: rgb(205, 219, 229); width: 443px; height: 417px;"
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border="1" cellpadding="2" cellspacing="0">
 +
  <tbody>
 +
    <tr>
 +
      <td><img style="width: 500px;" alt=""
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src="https://static.igem.org/mediawiki/2009/5/5e/Freiburg09_fokmodel_fig4.png" /><br />
 +
      </td>
 +
    </tr>
 +
    <tr>
 +
      <td><small>complete universal restriction enzyme.
 +
Blue: DNA strand; Red: two 16bp
 +
long Oligos,</small> <small>tags as indicated in the
 +
picture. Ochre: Fluorescein A
 +
binding lipocalin; Orange: digoxigenin</small> <small>binding
 +
lipocalin; Yellow:
 +
tagged Base with C6 linkers and attached tags; light Blue: inactive
 +
FokI</small> <small>cleavage domain; Red: FokI active
 +
cleavage domain; Green: three
 +
catalytically active aminoacids; White: two Calcium ions</small></td>
 +
    </tr>
 +
  </tbody>
 +
</table>
 +
<div style="text-align: justify;">The place where all the
 +
cutting events will take place in our scenery
 +
is the thermocycler. Therefore all the ingredients, i.e. chromosomal or
 +
plasmid DNA of interest, modified oligonucleotides and the different
 +
heterodimers Fok_a and Fok_i are mixed together. At high temperatures
 +
the DNA will denaturate allowing the different partners to find each
 +
other, cut the DNA, and fall apart in course of one thermocycle. The
 +
whole procedure can be repeated undefinately. To reach this step, we
 +
need to improve the thermostability of our enzyme via phage display.
 +
Creating a universal restriction enzyme provides not only the
 +
possibility to improve routine cloning but also to enhance therapeutic
 +
gene repair via triplex technology. Many genetic diseases and
 +
especially ones arising from single nucleotide polymorphisms (SNPs) or
 +
monogenetic disease can be alleviated by the replacement of mutated
 +
genes using this method. To cut double stranded DNA the
 +
oligonucleotides have to be replaced by triple helix forming oligos
 +
(TFO). They can bind double-stranded DNA in homopurin- or
 +
homopyrimidine-rich areas. But developments are also made to widen the
 +
possible interaction domains of the DNA and hence make the TFOs as
 +
programmable as our conventional oligonucleotides. In case of the human
 +
genome of 3&times;10^9 bp size, a highly specific artifical
 +
endonuclease
 +
would be necessary to address the mutated gene explicitly. The used
 +
TFOs therefore have to possess a minimum length of 16 bp to cut just
 +
once in the human genome (4^16 bp = 4.3*10^9 bp).<br />
 +
<br />
 +
Additionally, we provide an alternative way of binding by the coupling
 +
of the cleavage domain to the transcription factor Fos. This concept
 +
also includes the activity regulation by photo-switching. We are also
 +
modifying an argonaute protein into a DNA endonuclease using phage
 +
display technics.
 +
</div>
 +
</div>
</div>
</div>
 +
<br />
<br />
<div style="text-align: center;">
<div style="text-align: center;">

Revision as of 13:26, 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.








complete universal restriction enzyme. Blue: DNA strand; Red: two 16bp long Oligos, tags as indicated in the picture. Ochre: Fluorescein A binding lipocalin; Orange: digoxigenin binding lipocalin; Yellow: tagged Base with C6 linkers and attached tags; light Blue: inactive FokI cleavage domain; Red: FokI active cleavage domain; Green: three catalytically active aminoacids; White: two Calcium ions
The place where all the cutting events will take place in our scenery is the thermocycler. Therefore all the ingredients, i.e. chromosomal or plasmid DNA of interest, modified oligonucleotides and the different heterodimers Fok_a and Fok_i are mixed together. At high temperatures the DNA will denaturate allowing the different partners to find each other, cut the DNA, and fall apart in course of one thermocycle. The whole procedure can be repeated undefinately. To reach this step, we need to improve the thermostability of our enzyme via phage display. Creating a universal restriction enzyme provides not only the possibility to improve routine cloning but also to enhance therapeutic gene repair via triplex technology. Many genetic diseases and especially ones arising from single nucleotide polymorphisms (SNPs) or monogenetic disease can be alleviated by the replacement of mutated genes using this method. To cut double stranded DNA the oligonucleotides have to be replaced by triple helix forming oligos (TFO). They can bind double-stranded DNA in homopurin- or homopyrimidine-rich areas. But developments are also made to widen the possible interaction domains of the DNA and hence make the TFOs as programmable as our conventional oligonucleotides. In case of the human genome of 3×10^9 bp size, a highly specific artifical endonuclease would be necessary to address the mutated gene explicitly. The used TFOs therefore have to possess a minimum length of 16 bp to cut just once in the human genome (4^16 bp = 4.3*10^9 bp).

Additionally, we provide an alternative way of binding by the coupling of the cleavage domain to the transcription factor Fos. This concept also includes the activity regulation by photo-switching. We are also modifying an argonaute protein into a DNA endonuclease using phage display technics.





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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