Team:Freiburg bioware/Project/fokmonomer

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<div style="text-align: left;">
<div style="text-align: left;">
<p class="MsoNormal" style="text-align: justify;"><b
<p class="MsoNormal" style="text-align: justify;"><b
-
  style=""><u>Introduction<o:p></o:p></u></b></p>
+
  style=""><u>Introduction<o:p></o:p></u></b>
-
<p class="MsoNormal" style="text-align: justify;"><o:p>&nbsp;</o:p></p>
+
<p class="MsoNormal" style="text-align: justify;"><o:p>&nbsp;</o:p></p></div>
-
<p class="MsoNormal" style="text-align: justify;"><span
+
<p style="text-align: justify;"><span
  style="" lang="EN-GB">In order to create a universal
  style="" lang="EN-GB">In order to create a universal
restriction
restriction
-
enzyme we followed several ideas and models. The first and most
+
enzyme, we followed several ideas and models. The first and most
promising idea
promising idea
-
we had was to create a Fok Monomer as it represents the optimal and
+
we had was to create a monomeric restriction enzyme as it represents the optimal and
easiest
easiest
model for a universal restriction enzyme<o:p></o:p></span></p>
model for a universal restriction enzyme<o:p></o:p></span></p>
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  style="" lang="EN-GB">If we are able to employ a
  style="" lang="EN-GB">If we are able to employ a
monomeric enzyme,
monomeric enzyme,
-
this protein would have a couple of advantages. Most importantly we
+
this protein would have a couple of advantages: Most importantly we
would no longer
would no longer
need two separate oligonucleotides to achieve specific binding and
need two separate oligonucleotides to achieve specific binding and
-
cutting of
+
cleavage of
-
the target DNA. Also only one protein has to be purified &ndash;
+
the target DNA. Also, only one protein has to be purified &ndash;
thus saving time and
thus saving time and
money. A scientific advantage is the option to optimize the monomer by
money. A scientific advantage is the option to optimize the monomer by
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display. Using this technology, we would have the chance to create a
display. Using this technology, we would have the chance to create a
thermostable, specific, universal restriction enzyme whose DNA binding
thermostable, specific, universal restriction enzyme whose DNA binding
-
activity
+
specificity
-
is only created by a single oligonucleotide.<o:p></o:p></span></p>
+
is simply created by a single oligonucleotide.<o:p></o:p></span></p>
<p class="MsoNormal" style="text-align: justify;"><u><span
<p class="MsoNormal" style="text-align: justify;"><u><span
  style="" lang="EN-GB"><o:p><span
  style="" lang="EN-GB"><o:p><span
  style="text-decoration: none;"></span></o:p></span></u><span
  style="text-decoration: none;"></span></o:p></span></u><span
-
  style="" lang="EN-GB">Our goal was firstly to create
+
  style="" lang="EN-GB">Our goal was first, to create
-
a Fok-monomer which
+
a Fok-monomer that
-
is able to cut DNA without a primary dimerization step and secondly to
+
is able to cut DNA without a primary dimerization step, and second, to
show
show
that our heterodimeric interface design works properly. To reach this,
that our heterodimeric interface design works properly. To reach this,
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</o:p></span></p>
</o:p></span></p>
<p class="MsoNormal" style="text-align: justify;"><span
<p class="MsoNormal" style="text-align: justify;"><span
-
  style="" lang="EN-GB"><o:p><br />
+
  style="" lang="EN-GB"><o:p></b><br />
</o:p></span><b style=""><u><span
</o:p></span><b style=""><u><span
  style="" lang="EN-GB">Results<o:p></o:p></span></u></b></p>
  style="" lang="EN-GB">Results<o:p></o:p></span></u></b></p>
<p class="MsoNormal" style="text-align: justify;"><u><span
<p class="MsoNormal" style="text-align: justify;"><u><span
-
  style="" lang="EN-GB">3D modelling and design goals<o:p></o:p></span></u></p>
+
  style="" lang="EN-GB">3D modeling and design goals<o:p></o:p></span></u></p></b>
<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;"
<table style="text-align: left; width: 208px; height: 225px;"
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     </tr>
     </tr>
     <tr>
     <tr>
-
       <td><span style="font-size: 10pt;" lang="EN-GB">Fig1.:
+
       <td><span style="font-size: 10pt;" lang="EN-GB">Fig. 1:
-
       <st1:place w:st="on"><st2:Sn w:st="on">Scheme</st2:Sn>
+
       <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>
       <st2:Sn w:st="on">I.</st2:Sn></st1:place>
-
lipocalin<span style="">&nbsp; </span>II.
+
lipocalin,<span style="">&nbsp; </span>II.
-
Foka/i (heterodimers) III.
+
Foka/Foki (heterodimers), III.
-
Linker<span style="">&nbsp; </span>IV. His-Tag</span></td>
+
Linker,<span style="">&nbsp; </span>IV. His-Tag</span></td>
     </tr>
     </tr>
   </tbody>
   </tbody>
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  style="font-size: 10pt;" lang="EN-GB"></span><span
  style="font-size: 10pt;" lang="EN-GB"></span><span
  style="" lang="EN-US">The necessary parts in the
  style="" lang="EN-US">The necessary parts in the
-
model of an
+
model of a
-
monomeric restriction encyme are: Tag-binding protein - Fok
+
monomeric restriction enzyme are: Tag-binding protein (lipocalin) - Fok
heterodimer1 -
heterodimer1 -
-
linker - Fok herterodimer2 (Fig1). <o:p></o:p></span></p>
+
linker - Fok heterodimer2 (Fig. 1). <o:p></o:p></span></p>
<p class="MsoNormal" style="text-align: justify;"><span
<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>&nbsp;</o:p></span><span
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     </tr>
     </tr>
     <tr>
     <tr>
-
       <td style="text-align: center;">
+
       <td style="text-align: left;" valign="top">
-
      <h1 style="margin: 0cm 0cm 0.0001pt;"><span
+
      HisTag (BBa_K157011)<br>
-
style="font-size: 10pt; font-weight: normal;">HisTag
+
       StrepTag(BBa_K157012)
-
(BBa_K157011)<o:p></o:p></span></h1>
+
-
       <h1 style="margin: 0cm 0cm 0.0001pt;"><span
+
-
style="font-size: 10pt; font-weight: normal;">StrepTag
+
-
(BBa_K157012)<o:p></o:p></span></h1>
+
       </td>
       </td>
-
       <td style="text-align: center;">
+
       <td style="text-align: left;" valign="top">
-
      <h1 style="margin: 0cm 0cm 0.0001pt;"><span
+
      FluA(BBa_K157004)<br>
-
style="font-size: 10pt; font-weight: normal;">FluA
+
       DigA(BBa_K243003)<br>
-
(BBa_K157004)<o:p></o:p></span></h1>
+
       Fos(BBa_K243027)
-
       <h1 style="margin: 0cm 0cm 0.0001pt;"><span
+
-
style="font-size: 10pt; font-weight: normal;" lang="EN-GB">DigA
+
-
(BBa_K243003)<o:p></o:p></span></h1>
+
-
       <h1 style="margin: 0cm 0cm 0.0001pt;"><span
+
-
style="font-size: 10pt; font-weight: normal;" lang="EN-GB">Fos
+
-
(BBa_K243027)<o:p></o:p></span></h1>
+
       </td>
       </td>
-
       <td style="text-align: center;">
+
       <td style="text-align: left;" valign="top">
-
      <h1 style="margin: 0cm 0cm 0.0001pt;"><span
+
      GSAT-Linker(BBa_K243029)<br>
-
style="font-size: 10pt; font-weight: normal;">GSAT-Linker
+
       SEGLinker(BBa_K243030)
-
(BBa_K243029)<o:p></o:p></span></h1>
+
-
       <h1 style="margin: 0cm 0cm 0.0001pt;"><span
+
-
style="font-size: 10pt; font-weight: normal;">SEGLinker
+
-
(BBa_K243030)<o:p></o:p></span></h1>
+
       </td>
       </td>
-
       <td style="text-align: center;">
+
       <td style="text-align: left;" valign="top">
-
      <h1 style="margin: 0cm 2.85pt 0.0001pt 0cm;"><span
+
      FokA (K243000)<br>
-
style="font-size: 10pt; font-weight: normal;">FokA (K243000)<o:p></o:p></span></h1>
+
       Foki (K243001)
-
       <h1 style="margin: 0cm 2.85pt 0.0001pt 0cm;"><span
+
-
style="font-size: 10pt; font-weight: normal;">Foki (K243001)<o:p></o:p></span></h1>
+
       </td>
       </td>
     </tr>
     </tr>
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</o:p></span><span style="font-size: 10pt;"
</o:p></span><span style="font-size: 10pt;"
  lang="EN-US"><o:p></o:p></span><span
  lang="EN-US"><o:p></o:p></span><span
-
  style="font-size: 9pt;" lang="EN-US">Tab.:1 list of
+
  style="font-size: 9pt;" lang="EN-US">Tab. 1: List of
parts usable for
parts usable for
Fok Monomer<o:p></o:p></span></p>
Fok Monomer<o:p></o:p></span></p>
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  style="font-size: 9pt;" lang="EN-US"><o:p></o:p></span><span
  style="font-size: 9pt;" lang="EN-US"><o:p></o:p></span><span
  style="" lang="EN-US">In order to purify this fusion
  style="" lang="EN-US">In order to purify this fusion
-
protein we had
+
protein, we employed an N-terminal tag. The binding protein (a lipocalin-derived binding protein referred to as anticalin in analogy to antibodies) mediates  
-
to employ a N-terminal tag. The binding protein (a lipocalin) mediates
+
between the
between the
-
FokI protein and the tagged oligo</span><span style=""
+
FokI protein and the tagged oligonucleotide</span><span style=""
  lang="EN-GB">. Both parts of the Fok heterodimers are
  lang="EN-GB">. Both parts of the Fok heterodimers are
-
associated with a long linker allowing
+
associated with a long flexible linker allowing
them to establish the cutting site conformation. This artificial
them to establish the cutting site conformation. This artificial
restriction
restriction
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<p class="MsoNormal" style="text-align: justify;"><span
<p class="MsoNormal" style="text-align: justify;"><span
  style="" lang="EN-GB"><o:p></o:p>Before
  style="" lang="EN-GB"><o:p></o:p>Before
-
we started in the wet lab we planned all
+
we started in the wet lab, we thoroughly planned all
-
the individual steps and to figure out the best way to create the Fok
+
the individual steps. To figure out the best way to create the Fok
-
monomer,
+
monomer, we first designed it <i style="">in
-
so the first thing we did was to design a Fok monomer <i style="">in
+
silico.<o:p></o:p></i></span></p>
silico.<o:p></o:p></i></span></p>
<p class="MsoNormal" style="text-align: justify;"><span
<p class="MsoNormal" style="text-align: justify;"><span
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       <td><span style="" lang="EN-GB"><img
       <td><span style="" lang="EN-GB"><img
  style="width: 600px; height: 400px;" alt=""
  style="width: 600px; height: 400px;" alt=""
-
  src="https://static.igem.org/mediawiki/2009/e/ed/Freiburg09_Model1.JPG" /><br />
+
  src="https://static.igem.org/mediawiki/2009/1/15/Freiburg09_model1.png" /><br />
       </span>
       </span>
</td>
</td>
     </tr>
     </tr>
     <tr>
     <tr>
-
       <td><span style="font-size: 10pt;" lang="EN-GB">Fig2.:
+
       <td><span style="font-size: 10pt;" lang="EN-GB">Fig. 2:
3D model of Fok Monomer</span></td>
3D model of Fok Monomer</span></td>
     </tr>
     </tr>
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</table>
</table>
<p class="MsoNormal" style="text-align: justify;"><span
<p class="MsoNormal" style="text-align: justify;"><span
-
  style="" lang="EN-GB">The model (Fig2.) shows that we
+
  style="" lang="EN-GB">The model (Fig. 2) shows that we
have to use a
have to use a
-
linker which is something about 70 Angstr&ouml;m in length to
+
linker which is about 70 Angstrom in length to
-
provide the required
+
span the required
-
distance between the two Fok parts. Therefore we decided to order two
+
distance between the two Fok parts. Therefore, we decided to order two
-
complement oligonucleotides encoding one 36 amino acid glycine-serine
+
complementary oligonucleotides encoding a 36 amino acid long glycine-serine
linker.<o:p></o:p></span></p>
linker.<o:p></o:p></span></p>
<p class="MsoNormal" style="text-align: justify;"><span
<p class="MsoNormal" style="text-align: justify;"><span
  style="display:block; width:100%; overflow:scroll;" lang="EN-GB"><o:p>&nbsp;</o:p>
  style="display:block; width:100%; overflow:scroll;" lang="EN-GB"><o:p>&nbsp;</o:p>
-
5'-CTAGATGGCCGGCGGTTCTGGTGGTGGTTCTGGCGGTGGTTCTGGAGGTAGTTCTGGCGGTGGATCTGGAGGCGGTTCTGGGTCAGGATCTGGTGGAGGTTCTGGCTCTGGGAATCAGA-3'</br>
+
5'-CTAGATGGCCGGCGGTTCTGGTGGTGGTTCTGGCGGTGGTTCTGGAGGTAGTTCTGGCGGTGGATCTGGAGGCGGTTCTGGGTCAGGATCTGGTG<b
-
&nbsp;&nbsp;&nbsp;&nbsp;3'-TACCGGCCGCCAAGACCACCACCAAGACCGCCACCAAGACCTCCATCAAGACCGCCACCTAGACCTCCGCCAAGACCCAGTCCTAGACCAC<b
+
  style=""><span style="color: lime;">GA</span></b>GGTTCTGGCTCTGGGAATCAGA-3'</br>
-
  style=""><span style="color: red;">C</span></b>T<b
+
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3'-TACCGGCCGCCAAGACCACCACCAAGACCGCCACCAAGACCTCCATCAAGACCGCCACCTAGACCTCCGCCAAGACCCAGTCCTAGACCAC<b
-
  style=""><span style="color: lime;">A</span></b>CCAAGACCGAGACCCTTAGTCTGGCC-5<o:p></o:p></span></p>
+
  style=""><span style="color: lime;">TA</span></b>CCAAGACCGAGACCCTTAGTCTGGCC-5<o:p></o:p></span></p>
<p class="MsoNormal" style="text-align: justify;"><span
<p class="MsoNormal" style="text-align: justify;"><span
  style="" lang="EN-GB"><o:p></o:p><span
  style="" lang="EN-GB"><o:p></o:p><span
Line 310: Line 290:
  style="text-decoration: underline;">Cloning</span></span></p>
  style="text-decoration: underline;">Cloning</span></span></p>
<p class="MsoNormal" style="text-align: justify;"><span
<p class="MsoNormal" style="text-align: justify;"><span
-
  style="" lang="EN-GB">However at this stage the first
+
  style="" lang="EN-GB">However, at this stage the first
problem
problem
-
appeared.<o:p></o:p> This are the oligonucleotides, each
+
appeared.<o:p></o:p> These are the oligonucleotides, each
-
120aa long
+
120 nt long, which we ordered from Mr.Gene. Unfortunately, we made a mistake when
-
that we ordered from Mr.Gene. Unfortunately we made a mistake when
+
ordering the
ordering the
-
complement oligonucleotide. As you can see we have two mismatches
+
complementary oligonucleotide. Accidentally we introduced two mismatches
(compare
(compare
-
coloured bases in the sequence). But somehow we managed to dimerize the
+
colored bases in the sequence). But somehow we managed to dimerize the
two
two
-
oligos.<o:p></o:p> After dimerization in the thermo cycler
+
oligonucleotides.<o:p></o:p> After dimerization in the thermocycler
and
and
cloning into a pMA (BBa_K243031) vector (XbaI/AgeI) some mutations
cloning into a pMA (BBa_K243031) vector (XbaI/AgeI) some mutations
appeared in
appeared in
-
the 36GSLinker gene caused by the mismatches. But since there was no
+
the 36GSLinker gene caused by the mismatches. As there was no
frame
frame
shift we decided to carry on. <o:p></o:p><br />
shift we decided to carry on. <o:p></o:p><br />
-
Firstly we picked the parts we were going to
+
First, we picked the parts we were going to
use. We decided to use one we already completed in our earlier
use. We decided to use one we already completed in our earlier
-
experiments. His
+
experiments: His
&ndash; FluA &ndash; <st1:place w:st="on"><st1:City
&ndash; FluA &ndash; <st1:place w:st="on"><st1:City
  w:st="on">Split</st1:City></st1:place> -
  w:st="on">Split</st1:City></st1:place> -
-
Foki (BBa_K243010) has all the functions we needed. These are (I) a tag
+
Foki (BBa_K243010) had all the functions we needed. These are (i) a purification tag,  
-
to
+
(ii) a Fok domain and (iii) the anticalin that binds the
-
purify, (II) a Fok domain and (III) the anticalin which binds the
+
tagged
-
modified
+
oligonucleotide (Fig. 1). Cloning the linker (no part) behind these parts was
-
oligonucleotide Fig.1. Cloning the linker (no part) behind those was
+
the next
the next
-
step. We followed the assembly standard 25 and cut the vector with AgeI
+
step. We followed the assembly standard 25 and opened the vector with AgeI
and
and
-
PstI and the inserted with NgoMIV and PstI.<o:p></o:p></span></p>
+
PstI and cut the inserted with NgoMIV and PstI.<o:p></o:p></span></p>
<p class="MsoNormal" style="text-align: justify;"><span
<p class="MsoNormal" style="text-align: justify;"><span
  style="" lang="EN-GB"><o:p>
  style="" lang="EN-GB"><o:p>
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       <td>
       <td>
       <p class="MsoNormal"><span
       <p class="MsoNormal"><span
-
  style="font-size: 10pt;" lang="EN-GB">Fig3. gele
+
  style="font-size: 10pt;" lang="EN-GB">Fig. 3: Gel
picture of test digest<o:p></o:p></span></p>
picture of test digest<o:p></o:p></span></p>
       </td>
       </td>
Line 362: Line 340:
</o:p><br />
</o:p><br />
However, we ran into some more problems. After cloning Foka
However, we ran into some more problems. After cloning Foka
-
(BBa_K243000) behind this construct, again assembly standard 25, we
+
(BBa_K243000) after the other parts, again assembly standard 25, we
-
performed
+
performed test restriction digests cutting out the insert to confirm its size. These digests showed that the
-
some test digestions to cut out the insert again. These showed that the
+
plasmid
plasmid
-
became even smaller as it would be without an insertion (pEX: ~ 4,5kb
+
became even smaller as it would be without an insertion (pEX: ~ 4,5kbp
and after
and after
-
test digestion ~ 2,2kb). The Insert should be about 1,9kb but as you
+
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 />
-
can see in
+
-
Fig.3 it&acute;s about 900k. <span style="">&nbsp;</span>Also
+
-
the sequencing
+
-
went wrong.<o:p><br />
+
</o:p></span></p>
</o:p></span></p>
<p class="MsoNormal" style="text-align: justify;"><span
<p class="MsoNormal" style="text-align: justify;"><span
-
  style="" lang="EN-GB">Most likely the active Fok did
+
  style="" lang="EN-GB">Most likely, the active Fok did
cut the plasmid
cut the plasmid
-
and promoted deletion of the Fok gene, what gave mutated cells a
+
and promoted deletion of the Fok gene, which gave such mutated cells a
significant
significant
-
growth advantage. After that we decided to order the 36GS-linker (GSAT
+
growth advantage. After we encountered these problems, we decided to order the 36GS-linker (GSAT
Linker:
Linker:
BBa_K243029) as gene synthesis.<o:p></o:p></span></p>
BBa_K243029) as gene synthesis.<o:p></o:p></span></p>
Line 387: Line 360:
<p class="MsoNormal" style="text-align: justify;"><span
<p class="MsoNormal" style="text-align: justify;"><span
  style="" lang="EN-GB"><o:p></o:p>This
  style="" lang="EN-GB"><o:p></o:p>This
-
time the order was well planned but it
+
time the order was well planned but, unfortunately, it
-
took over 4 weeks to get them. Unfortunately the genes arrived too
+
took over 4 weeks for the synthesis and delivery, and the genes arrived too
late. Once
late. Once
-
again we have to make all the cloning steps. As of mid October 2009 the
+
again we have to make all the cloning steps. As of mid October 2009, the
project
project
is still in progress.<o:p></o:p></span></p>
is still in progress.<o:p></o:p></span></p>

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.