Team:Freiburg bioware/Project/fokmonomer

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<div class="art-PostContent">
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<div style="text-align: left;">
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<p class="MsoNormal" style="text-align: center;"
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align="center"><b><span
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style="font-size: 14pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;">planning and cloning a monomeric restriction enzyme
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<br />
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A Survey by the &ldquo;international Genetically Engineered
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Machine&rdquo; <o:p></o:p></span></b></p>
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<p class="MsoNormal" style="text-align: center;"
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align="center"><b><span
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style="font-size: 14pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;">(iGEM)
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Team Freiburg 2009</span></b><b style=""><span
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style="font-size: 14pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"><o:p></o:p></span></b></p>
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<p class="MsoNormal" style="text-align: justify;"><b
<p class="MsoNormal" style="text-align: justify;"><b
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  style=""><span style=""><o:p>&nbsp;</o:p></span></b></p>
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  style=""><u>Introduction<o:p></o:p></u></b>
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<p class="MsoNormal" style="text-align: justify;"><b
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<p class="MsoNormal" style="text-align: justify;"><o:p>&nbsp;</o:p></p></div>
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style=""><span style=""><o:p>&nbsp;</o:p></span></b></p>
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<p style="text-align: justify;"><span
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<p class="MsoNormal" style="text-align: justify;"><b
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  style="" lang="EN-GB">In order to create a universal
-
  style=""><span
+
restriction
-
style="font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;">Introduction<o:p></o:p></span></b></p>
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enzyme, we followed several ideas and models. The first and most
-
<p class="MsoNormal" style="text-align: justify;"><b
+
promising idea
-
  style=""><span
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we had was to create a monomeric restriction enzyme as it represents the optimal and
-
  style="font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;">
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easiest
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model for a universal restriction enzyme<o:p></o:p></span></p>
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Introduction<br><br>
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<p class="MsoNormal" style="text-align: justify;"><u><span
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  style="" lang="EN-GB"><o:p><span
-
In order to create a universal restriction enzyme we followed several ideas and models. The first and most promising idea we had was to create a Fok Monomer as it represents the optimal and easiest model for a universal restriction enzyme<br><br>
+
  style="text-decoration: none;"></span></o:p></span></u><span
-
 
+
style="" lang="EN-GB">If we are able to employ a
-
If we are able to employ a monomeric enzyme, this protein would have a couple of advantages. Most importantly we would no longer need two separate oligonucleotides to achieve specific binding and cutting of the target DNA. Also only one protein has to be purified thus saving time and money. A scientific advantage is the option to optimize the monomer by phage display. Using this technology, we would have the chance to create a thermostable, specific, universal restriction enzyme whose DNA binding activity is only created by a single oligonucleotide.<br><br>
+
monomeric enzyme,
-
 
+
this protein would have a couple of advantages: Most importantly we
-
Our goal was firstly to create a Fok-monomer which is able to cut DNA without a primary dimerization step and secondly to show that our heterodimeric interface design works properly. To reach this, we had to clone all the required parts one after another, resulting in a huge fusion protein.<br><br>
+
would no longer
-
 
+
need two separate oligonucleotides to achieve specific binding and
-
 
+
cleavage of
-
Results<br><br>
+
the target DNA. Also, only one protein has to be purified &ndash;
-
 
+
thus saving time and
-
3D modelling and design goals<br><br>
+
money. A scientific advantage is the option to optimize the monomer by
-
 
+
phage
-
   
+
display. Using this technology, we would have the chance to create a
-
Fig1.: Scheme I. lipocalin  II. Foka/i (heterodimers) III. Linker  IV. His-Tag<br><br>
+
thermostable, specific, universal restriction enzyme whose DNA binding
-
 
+
specificity
-
The necessary parts in the model of an monomeric restriction encyme are: Tag-binding protein - Fok heterodimer1 - linker - Fok herterodimer2 (Fig1). <br><br>
+
is simply created by a single oligonucleotide.<o:p></o:p></span></p>
-
 
+
<p class="MsoNormal" style="text-align: justify;"><u><span
-
+
style="" lang="EN-GB"><o:p><span
-
Tab.:1 list of parts usable for Fok Monomer<br><br>
+
style="text-decoration: none;"></span></o:p></span></u><span
-
 
+
style="" lang="EN-GB">Our goal was first, to create
-
In order to purify this fusion protein we had to employ a N-terminal tag. The binding protein (a lipocalin) mediates between the FokI protein and the tagged oligo. Both parts of the Fok heterodimers are associated with a long linker allowing them to establish the cutting site conformation. This artificial restriction nuclease domain is the functional part of the monomer and catalyzes the DNA cleavage.<br><br>
+
a Fok-monomer that
-
 
+
is able to cut DNA without a primary dimerization step, and second, to
-
Before we started in the wet lab we planned all the individual steps and to figure out the best way to create the Fok monomer, so the first thing we did was to design a Fok monomer in silico.<br><br>
+
show
-
   
+
that our heterodimeric interface design works properly. To reach this,
-
+
we had
-
Fig2.: 3D model of Fok Monomer<br><br>
+
to clone all the required parts one after another, resulting in a huge
-
 
+
fusion
-
The model (Fig2.) shows that we have to use a linker which is something about 70 Angström in length to provide the required distance between the two Fok parts. Therefore we decided to order two complement oligonucleotides encoding one 36 amino acid glycine-serine linker.<br><br>
+
protein.<o:p><br />
-
 
+
</o:p></span></p>
-
5'-CTAGATGGCCGGCGGTTCTGGTGGTGGTTCTGGCGGTGGTTCTGGAGGTAGTTCTGGCGGTGGATCTGGAGGCGGTTCTGGGTCAGGATCTGGTGGAGGTTCTGGCTCTGGGAATCAGA-3'<br>
+
<p class="MsoNormal" style="text-align: justify;"><span
-
3'-TACCGGCCGCCAAGACCACCACCAAGACCGCCACCAAGACCTCCATCAAGACCGCCACCTAGACCTCCGCCAAGACCCAGTCCTAGACCACCTACCAAGACCGAGACCCTTAGTCTGGCC-5<br>
+
  style="" lang="EN-GB"><o:p></b><br />
-
 
+
</o:p></span><b style=""><u><span
-
However at this stage the first problem appeared.<br>
+
  style="" lang="EN-GB">Results<o:p></o:p></span></u></b></p>
-
This are the oligonucleotides, each 120aa long that we ordered from Mr.Gene. Unfortunately we made a mistake when ordering the complement oligonucleotide. As you can see we have two mismatches (compare coloured bases in the sequence). But somehow we managed to dimerize the two oligos.<br>
+
<p class="MsoNormal" style="text-align: justify;"><u><span
-
After dimerization in the thermo cycler and cloning into a pMA (BBa_K243031) vector (XbaI/AgeI) some mutations appeared in the 36GSLinker gene caused by the mismatches. But since there was no frame shift we decided to carry on.
+
  style="" lang="EN-GB">3D modeling and design goals<o:p></o:p></span></u></p></b>
-
Firstly we picked the parts we were going to use. We decided to use one we already completed in our earlier experiments. His – FluA – Split - Foki (BBa_K243010) has all the functions we needed. These are (I) a tag to purify, (II) a Fok domain and (III) the anticalin which binds the modified oligonucleotide Fig.1. Cloning the linker (no part) behind those was the next step. We followed the assembly standard 25 and cut the vector with AgeI and PstI and the inserted with NgoMIV and PstI.<br><br><br>
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
However, we ran into some more problems. After cloning Foka (BBa_K243000) behind this construct, again assembly standard 25, we performed some test digestions to cut out the insert again. These showed that the plasmid became even smaller as it would be without an insertion (pEX: ~ 4,5kb and after test digestion ~ 2,2kb). The Insert should be about 1,9kb but as you can see in Fig.3 it´s about 900k. Also the sequencing went wrong.<br><br><br>
+
-
 
+
-
 
+
-
 
+
-
Most likely the active Fok did cut the plasmid and promoted deletion of the Fok gene, what gave mutated cells a significant growth advantage. After that we decided to order the 36GS-linker (GSAT Linker: BBa_K243029) as gene synthesis.<br><br>
+
-
 
+
-
5´-GAGCTCGAATTCGCGGCCGCTTCTAGATGGCCGGCGGTGGTTCTGCCGGT
+
-
GGCTCCGGTTCTGGCTCCAGCGGTGGCAGCTCTGGTGCGTCCGGCACGGG
+
-
TACTGCGGGTGGCACTGGCAGCGGTTCCGGTACTGGCTCTGGCACCGGTA
+
-
ATACTAGTAGCGGCCGCTGCAGGGTACC-3´<br><br>
+
-
 
+
-
This time the order was well planned but it took over 4 weeks to get them. Unfortunately the genes arrived too late. Once again we have to make all the cloning steps. As of mid October 2009 the project is still in progress.
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
</div>
+
<p class="MsoNormal" style="text-align: justify;"></p>
<p class="MsoNormal" style="text-align: justify;"></p>
 +
<table style="text-align: left; width: 208px; height: 225px;"
 +
border="1" cellpadding="0" cellspacing="0">
 +
  <tbody>
 +
    <tr>
 +
      <td><img style="width: 600px; height: 300px;"
 +
alt=""
 +
src="https://static.igem.org/mediawiki/2009/2/29/Freiburg09_Monomermodel1.JPG" /></td>
 +
    </tr>
 +
    <tr>
 +
      <td><span style="font-size: 10pt;" lang="EN-GB">Fig. 1:
 +
      <st1:place w:st="on"><st2:Sn w:st="on">Scheme of monomoeric Fok:</st2:Sn>
 +
      <st2:Sn w:st="on">I.</st2:Sn></st1:place>
 +
lipocalin,<span style="">&nbsp; </span>II.
 +
Foka/Foki (heterodimers), III.
 +
Linker,<span style="">&nbsp; </span>IV. His-Tag</span></td>
 +
    </tr>
 +
  </tbody>
 +
</table>
<p class="MsoNormal" style="text-align: justify;"><span
<p class="MsoNormal" style="text-align: justify;"><span
-
  style="font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"><o:p></o:p></span></p>
+
style="" lang="EN-GB"><o:p></o:p></span><span
 +
  style="font-size: 10pt;" lang="EN-GB"></span><span
 +
style="" lang="EN-US">The necessary parts in the
 +
model of a
 +
monomeric restriction enzyme are: Tag-binding protein (lipocalin) - Fok
 +
heterodimer1 -
 +
linker - Fok heterodimer2 (Fig. 1). <o:p></o:p></span></p>
 +
<p class="MsoNormal" style="text-align: justify;"><span
 +
style="font-size: 10pt;" lang="EN-US"><o:p>&nbsp;</o:p></span><span
 +
style="font-size: 10pt;" lang="EN-US"><o:p>
 +
<table style="text-align: left; width: 705px; height: 50px;"
 +
border="1" cellpadding="0" cellspacing="0">
 +
  <tbody>
 +
    <tr>
 +
      <td style="text-align: center;">Tag</td>
 +
      <td style="text-align: center;">binding protein</td>
 +
      <td style="text-align: center;">linker</td>
 +
      <td style="text-align: center;">Fok heterodimer</td>
 +
    </tr>
 +
    <tr>
 +
      <td style="text-align: left;" valign="top">
 +
      HisTag (BBa_K157011)<br>
 +
      StrepTag(BBa_K157012)
 +
      </td>
 +
      <td style="text-align: left;" valign="top">
 +
      FluA(BBa_K157004)<br>
 +
      DigA(BBa_K243003)<br>
 +
      Fos(BBa_K243027)
 +
      </td>
 +
      <td style="text-align: left;" valign="top">
 +
      GSAT-Linker(BBa_K243029)<br>
 +
      SEGLinker(BBa_K243030)
 +
      </td>
 +
      <td style="text-align: left;" valign="top">
 +
      FokA (K243000)<br>
 +
      Foki (K243001)
 +
      </td>
 +
    </tr>
 +
  </tbody>
 +
</table>
 +
</o:p></span><span style="font-size: 10pt;"
 +
lang="EN-US"><o:p></o:p></span><span
 +
style="font-size: 9pt;" lang="EN-US">Tab. 1: List of
 +
parts usable for
 +
Fok Monomer<o:p></o:p></span></p>
 +
<p class="MsoNormal" style="text-align: justify;"><span
 +
style="font-size: 9pt;" lang="EN-US"><o:p></o:p></span><span
 +
style="" lang="EN-US">In order to purify this fusion
 +
protein, we employed an N-terminal tag. The binding protein (a lipocalin-derived binding protein referred to as anticalin in analogy to antibodies) mediates
 +
between the
 +
FokI protein and the tagged oligonucleotide</span><span style=""
 +
lang="EN-GB">. Both parts of the Fok heterodimers are
 +
associated with a long flexible linker allowing
 +
them to establish the cutting site conformation. This artificial
 +
restriction
 +
nuclease domain is the functional part of the monomer and catalyzes the
 +
DNA
 +
cleavage.<o:p></o:p></span></p>
 +
<p class="MsoNormal" style="text-align: justify;"><span
 +
style="" lang="EN-GB"><o:p></o:p>Before
 +
we started in the wet lab, we thoroughly planned all
 +
the individual steps. To figure out the best way to create the Fok
 +
monomer, we first designed it <i style="">in
 +
silico.<o:p></o:p></i></span></p>
 +
<p class="MsoNormal" style="text-align: justify;"><span
 +
style="" lang="EN-GB"><span style=""></span></span></p>
 +
<table style="text-align: left; width: 196px; height: 225px;"
 +
border="1" cellpadding="0" cellspacing="0">
 +
  <tbody>
 +
    <tr>
 +
      <td><span style="" lang="EN-GB"><img
 +
style="width: 600px; height: 400px;" alt=""
 +
src="https://static.igem.org/mediawiki/2009/1/15/Freiburg09_model1.png" /><br />
 +
      </span>
 +
</td>
 +
    </tr>
 +
    <tr>
 +
      <td><span style="font-size: 10pt;" lang="EN-GB">Fig. 2:
 +
3D model of Fok Monomer</span></td>
 +
    </tr>
 +
  </tbody>
 +
</table>
 +
<p class="MsoNormal" style="text-align: justify;"><span
 +
style="" lang="EN-GB">The model (Fig. 2) shows that we
 +
have to use a
 +
linker which is about 70 Angstrom in length to
 +
span the required
 +
distance between the two Fok parts. Therefore, we decided to order two
 +
complementary oligonucleotides encoding a 36 amino acid long glycine-serine
 +
linker.<o:p></o:p></span></p>
 +
<p class="MsoNormal" style="text-align: justify;"><span
 +
style="display:block; width:100%; overflow:scroll;" lang="EN-GB"><o:p>&nbsp;</o:p>
 +
5'-CTAGATGGCCGGCGGTTCTGGTGGTGGTTCTGGCGGTGGTTCTGGAGGTAGTTCTGGCGGTGGATCTGGAGGCGGTTCTGGGTCAGGATCTGGTG<b
 +
style=""><span style="color: lime;">GA</span></b>GGTTCTGGCTCTGGGAATCAGA-3'</br>
 +
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3'-TACCGGCCGCCAAGACCACCACCAAGACCGCCACCAAGACCTCCATCAAGACCGCCACCTAGACCTCCGCCAAGACCCAGTCCTAGACCAC<b
 +
style=""><span style="color: lime;">TA</span></b>CCAAGACCGAGACCCTTAGTCTGGCC-5<o:p></o:p></span></p>
 +
<p class="MsoNormal" style="text-align: justify;"><span
 +
style="" lang="EN-GB"><o:p></o:p><span
 +
style="text-decoration: underline;"></span></span></p>
 +
<p class="MsoNormal" style="text-align: justify;"><span
 +
style="" lang="EN-GB"><span
 +
style="text-decoration: underline;">Cloning</span></span></p>
 +
<p class="MsoNormal" style="text-align: justify;"><span
 +
style="" lang="EN-GB">However, at this stage the first
 +
problem
 +
appeared.<o:p></o:p> These are the oligonucleotides, each
 +
120 nt long, which we ordered from Mr.Gene. Unfortunately, we made a mistake when
 +
ordering the
 +
complementary oligonucleotide. Accidentally we introduced two mismatches
 +
(compare
 +
colored bases in the sequence). But somehow we managed to dimerize the
 +
two
 +
oligonucleotides.<o:p></o:p> After dimerization in the thermocycler
 +
and
 +
cloning into a pMA (BBa_K243031) vector (XbaI/AgeI) some mutations
 +
appeared in
 +
the 36GSLinker gene caused by the mismatches. As there was no
 +
frame
 +
shift we decided to carry on. <o:p></o:p><br />
 +
First, we picked the parts we were going to
 +
use. We decided to use one we already completed in our earlier
 +
experiments: His
 +
&ndash; FluA &ndash; <st1:place w:st="on"><st1:City
 +
w:st="on">Split</st1:City></st1:place> -
 +
Foki (BBa_K243010) had all the functions we needed. These are (i) a purification tag,
 +
(ii) a Fok domain and (iii) the anticalin that binds the
 +
tagged
 +
oligonucleotide (Fig. 1). Cloning the linker (no part) behind these parts was
 +
the next
 +
step. We followed the assembly standard 25 and opened the vector with AgeI
 +
and
 +
PstI and cut the inserted with NgoMIV and PstI.<o:p></o:p></span></p>
 +
<p class="MsoNormal" style="text-align: justify;"><span
 +
style="" lang="EN-GB"><o:p>
 +
<table style="text-align: left; width: 217px; height: 249px;"
 +
border="1" cellpadding="0" cellspacing="0">
 +
  <tbody>
 +
    <tr>
 +
      <td><img style="width: 490px; height: 400px;"
 +
alt=""
 +
src="https://static.igem.org/mediawiki/2009/thumb/b/bd/Freiburg09_TestverdauFokM1.JPG/792px-Freiburg09_TestverdauFokM1.JPG" /></td>
 +
    </tr>
 +
    <tr>
 +
      <td>
 +
      <p class="MsoNormal"><span
 +
style="font-size: 10pt;" lang="EN-GB">Fig. 3: Gel
 +
picture of test digest<o:p></o:p></span></p>
 +
      </td>
 +
    </tr>
 +
  </tbody>
 +
</table>
 +
</o:p><br />
 +
However, we ran into some more problems. After cloning Foka
 +
(BBa_K243000) after the other parts, again assembly standard 25, we
 +
performed test restriction digests cutting out the insert to confirm its size. These digests showed that the
 +
plasmid
 +
became even smaller as it would be without an insertion (pEX: ~ 4,5kbp
 +
and after
 +
test digestion ~ 2,2kbp). The insert should have a size of about 1.9 kbp but was found to be only about 900 bp (Fig. 3). <span style="">&nbsp;</span>The sequencing did not work either.<o:p><br />
 +
</o:p></span></p>
 +
<p class="MsoNormal" style="text-align: justify;"><span
 +
style="" lang="EN-GB">Most likely, the active Fok did
 +
cut the plasmid
 +
and promoted deletion of the Fok gene, which gave such mutated cells a
 +
significant
 +
growth advantage. After we encountered these problems, we decided to order the 36GS-linker (GSAT
 +
Linker:
 +
BBa_K243029) as gene synthesis.<o:p></o:p></span></p>
 +
<p class="MsoNormal" style="text-align: justify;"><span
 +
style="" lang="EN-GB"><o:p>&nbsp;</o:p>5&acute;-GAGCTCGAATTCGCGGCCGCTTCTAGATGGCCGGCGGTGGTTCTGCCGGT<o:p></o:p>GGCTCCGGTTCTGGCTCCAGCGGTGGCAGCTCTGGTGCGTCCGGCACGGG<o:p></o:p><br />
 +
TACTGCGGGTGGCACTGGCAGCGGTTCCGGTACTGGCTCTGGCACCGGTA<o:p></o:p>ATACTAGTAGCGGCCGCTGCAGGGTACC-3&acute;<o:p></o:p></span></p>
 +
<p class="MsoNormal" style="text-align: justify;"><span
 +
style="" lang="EN-GB"><o:p></o:p>This
 +
time the order was well planned but, unfortunately, it
 +
took over 4 weeks for the synthesis and delivery, and the genes arrived too
 +
late. Once
 +
again we have to make all the cloning steps. As of mid October 2009, the
 +
project
 +
is still in progress.<o:p></o:p></span></p>
</div>
</div>
 +
<br />
 +
<div style="text-align: center;">
 +
</div>
 +
<br />
</div>
</div>
<div class="cleared"></div>
<div class="cleared"></div>

Latest revision as of 02:19, 22 October 2009

FREiGEM

Introduction

 

In order to create a universal restriction enzyme, we followed several ideas and models. The first and most promising idea we had was to create a monomeric restriction enzyme as it represents the optimal and easiest model for a universal restriction enzyme

If we are able to employ a monomeric enzyme, this protein would have a couple of advantages: Most importantly we would no longer need two separate oligonucleotides to achieve specific binding and cleavage of the target DNA. Also, only one protein has to be purified – thus saving time and money. A scientific advantage is the option to optimize the monomer by phage display. Using this technology, we would have the chance to create a thermostable, specific, universal restriction enzyme whose DNA binding specificity is simply created by a single oligonucleotide.

Our goal was first, to create a Fok-monomer that is able to cut DNA without a primary dimerization step, and second, to show that our heterodimeric interface design works properly. To reach this, we had to clone all the required parts one after another, resulting in a huge fusion protein.


Results

3D modeling and design goals

Fig. 1: Scheme of monomoeric Fok: I. lipocalin,  II. Foka/Foki (heterodimers), III. Linker,  IV. His-Tag

The necessary parts in the model of a monomeric restriction enzyme are: Tag-binding protein (lipocalin) - Fok heterodimer1 - linker - Fok heterodimer2 (Fig. 1).

 

Tag binding protein linker Fok heterodimer
HisTag (BBa_K157011)
StrepTag(BBa_K157012)
FluA(BBa_K157004)
DigA(BBa_K243003)
Fos(BBa_K243027)
GSAT-Linker(BBa_K243029)
SEGLinker(BBa_K243030)
FokA (K243000)
Foki (K243001)
Tab. 1: List of parts usable for Fok Monomer

In order to purify this fusion protein, we employed an N-terminal tag. The binding protein (a lipocalin-derived binding protein referred to as anticalin in analogy to antibodies) mediates between the FokI protein and the tagged oligonucleotide. Both parts of the Fok heterodimers are associated with a long flexible linker allowing them to establish the cutting site conformation. This artificial restriction nuclease domain is the functional part of the monomer and catalyzes the DNA cleavage.

Before we started in the wet lab, we thoroughly planned all the individual steps. To figure out the best way to create the Fok monomer, we first designed it in silico.


Fig. 2: 3D model of Fok Monomer

The model (Fig. 2) shows that we have to use a linker which is about 70 Angstrom in length to span the required distance between the two Fok parts. Therefore, we decided to order two complementary oligonucleotides encoding a 36 amino acid long glycine-serine linker.

  5'-CTAGATGGCCGGCGGTTCTGGTGGTGGTTCTGGCGGTGGTTCTGGAGGTAGTTCTGGCGGTGGATCTGGAGGCGGTTCTGGGTCAGGATCTGGTGGAGGTTCTGGCTCTGGGAATCAGA-3'
         3'-TACCGGCCGCCAAGACCACCACCAAGACCGCCACCAAGACCTCCATCAAGACCGCCACCTAGACCTCCGCCAAGACCCAGTCCTAGACCACTACCAAGACCGAGACCCTTAGTCTGGCC-5

Cloning

However, at this stage the first problem appeared. These are the oligonucleotides, each 120 nt long, which we ordered from Mr.Gene. Unfortunately, we made a mistake when ordering the complementary oligonucleotide. Accidentally we introduced two mismatches (compare colored bases in the sequence). But somehow we managed to dimerize the two oligonucleotides. After dimerization in the thermocycler and cloning into a pMA (BBa_K243031) vector (XbaI/AgeI) some mutations appeared in the 36GSLinker gene caused by the mismatches. As there was no frame shift we decided to carry on.
First, we picked the parts we were going to use. We decided to use one we already completed in our earlier experiments: His – FluA – Split - Foki (BBa_K243010) had all the functions we needed. These are (i) a purification tag, (ii) a Fok domain and (iii) the anticalin that binds the tagged oligonucleotide (Fig. 1). Cloning the linker (no part) behind these parts was the next step. We followed the assembly standard 25 and opened the vector with AgeI and PstI and cut the inserted with NgoMIV and PstI.

Fig. 3: Gel picture of test digest


However, we ran into some more problems. After cloning Foka (BBa_K243000) after the other parts, again assembly standard 25, we performed test restriction digests cutting out the insert to confirm its size. These digests showed that the plasmid became even smaller as it would be without an insertion (pEX: ~ 4,5kbp and after test digestion ~ 2,2kbp). The insert should have a size of about 1.9 kbp but was found to be only about 900 bp (Fig. 3).  The sequencing did not work either.

Most likely, the active Fok did cut the plasmid and promoted deletion of the Fok gene, which gave such mutated cells a significant growth advantage. After we encountered these problems, we decided to order the 36GS-linker (GSAT Linker: BBa_K243029) as gene synthesis.

 5´-GAGCTCGAATTCGCGGCCGCTTCTAGATGGCCGGCGGTGGTTCTGCCGGTGGCTCCGGTTCTGGCTCCAGCGGTGGCAGCTCTGGTGCGTCCGGCACGGG
TACTGCGGGTGGCACTGGCAGCGGTTCCGGTACTGGCTCTGGCACCGGTAATACTAGTAGCGGCCGCTGCAGGGTACC-3´

This time the order was well planned but, unfortunately, it took over 4 weeks for the synthesis and delivery, and the genes arrived too late. Once again we have to make all the cloning steps. As of mid October 2009, the project is still in progress.