Team:Freiburg Bioware/Project/Results/Modularization Vector Plasmid

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<html>
<html>
-
<h2>Modularization GOI/Vector Plasmid</h2>
+
<p class="MsoNormal">&nbsp;</p>
-
<h3>Introduction to Modularization</h3>
+
<p class="MsoTocHeading">Contents</p>
 +
<p class="MsoToc2"><span class="MsoHyperlink"><a
 +
href="#_Toc275794992">Modularization
 +
GOI/Vector Plasmid<span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">1</span></a></span></p>
 +
<p class="MsoToc3"><span class="MsoHyperlink"><a
 +
href="#_Toc275794993">Introduction
 +
to Modularization<span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">1</span></a></span></p>
 +
<p class="MsoToc3"><span class="MsoHyperlink"><a
 +
href="#_Toc275794994">Recombinant
 +
and Modular Vector Plasmid Carrying GOI<span
 +
style="color: windowtext; display: none; text-decoration: none;">
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">1</span></a></span></p>
 +
<p class="MsoToc3"><span class="MsoHyperlink"><a
 +
href="#_Toc275794995">Cloning and
 +
Combination Strategies for the Vectorplasmid<span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">2</span></a></span></p>
 +
<p class="MsoToc3"><span class="MsoHyperlink"><a
 +
href="#_Toc275794996">Testing
 +
functionality of Assembled Vectorplasmid<span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">3</span></a></span></p>
 +
<p class="MsoNormal">&nbsp;</p>
 +
<p class="MsoNormal">&nbsp;</p>
 +
<p class="MsoNormal">&nbsp;</p>
 +
<h2><a name="_Toc275794992">Modularization
 +
GOI/Vector Plasmid</a></h2>
 +
<h3><a name="_Toc275794993">Introduction to
 +
Modularization</a></h3>
<p class="MsoNormal">Producing recombinant virus particles
<p class="MsoNormal">Producing recombinant virus particles
for therapeutical
for therapeutical
means is, besides specifically target cells, purification and
means is, besides specifically target cells, purification and
-
quantification
+
quantification assays
-
assays of AAV-2, one intention of the Virus Construction Kit provided
+
of AAV-2, one intention of the Virus Construction Kit provided by the
-
by the
+
iGEM team
-
iGEM team Freiburg_Bioware 2010. For obtaining a modular toolkit, the
+
Freiburg_Bioware 2010. For obtaining a modular toolkit, the complex
-
complex
+
components
-
components of AAV-2 were extracted and redesigned to match the iGEM
+
of AAV-2 were extracted and redesigned to match the iGEM standard.
-
standard.
+
Functional
-
Functional activity was tested in cell culture.</p>
+
activity was tested in cell culture.</p>
<p class="MsoNormal">Differing from the wildtype AAV-2
<p class="MsoNormal">Differing from the wildtype AAV-2
genome, the Helper Free
genome, the Helper Free
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express the
express the
stably integrated E1A and E1B helper proteins for efficient virus
stably integrated E1A and E1B helper proteins for efficient virus
-
production. The
+
production.
-
plasmid containing the inverted terminal repeats (ITRs) is encapsidated
+
The plasmid containing the inverted terminal repeats (ITRs) is
-
into
+
encapsidated
-
the preformed capsids after production of single-stranded DNA therefore
+
into the preformed capsids after production of single-stranded DNA
-
also
+
therefore
-
known as vectorplasmid (pGOI). Promoter, <i>beta-globin</i>
+
also known as vectorplasmid (pGOI). Promoter, <i>beta-globin</i>
-
intron and the hGH
+
intron and the
-
terminator signal are flanked by the ITRs and serve in the host cell
+
hGH terminator signal are flanked by the ITRs and serve in the host
-
for
+
cell for
regulation of transgene expression. In addition to that, the plasmid
regulation of transgene expression. In addition to that, the plasmid
coding for
coding for
the Rep and Cap proteins (pRC) can be provided <i>in trans</i>
the Rep and Cap proteins (pRC) can be provided <i>in trans</i>
-
leading to a
+
leading to a layer
-
layer of specificity due to the fact that the two genes are not
+
of specificity due to the fact that the two genes are not packaged into
-
packaged into
+
the
-
the capsid since lacking of the ITRs impairs encapsidation. Another
+
capsid since lacking of the ITRs impairs encapsidation. Another
-
advantage
+
advantage of
-
of the Helper Free System can be attributed to cotransfection of
+
the Helper Free System can be attributed to cotransfection of another
-
another helper
+
helper
plasmid (pHelper), which provides the necessary proteins normally
plasmid (pHelper), which provides the necessary proteins normally
obtained by
obtained by
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gene
gene
expression of Rep and Cap proteins.</p>
expression of Rep and Cap proteins.</p>
-
<h3>Recombinant and Modular Vector Plasmid Carrying GOI</h3>
+
<h3><a name="_Toc275794994">Recombinant and Modular
 +
Vector Plasmid Carrying GOI</a></h3>
<p class="MsoNormal">The iGEM team Freiburg_Bioware 2010
<p class="MsoNormal">The iGEM team Freiburg_Bioware 2010
provides a modular Virus
provides a modular Virus
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and
and
purification approaches depending on capsid modifications and the gene
purification approaches depending on capsid modifications and the gene
-
of
+
of interest
-
interest flanked by the inverted terminal repeats (ITRs. In order to
+
flanked by the inverted terminal repeats (ITRs. In order to produce
-
produce
+
BioBrick-compatible standardized biological parts, we reengineered the
BioBrick-compatible standardized biological parts, we reengineered the
plasmids
plasmids
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BBa_K404101), a
BBa_K404101), a
promoter (pCMV :BBa_K404102 or phTERT: BBa_K404106)) , the beta-globin
promoter (pCMV :BBa_K404102 or phTERT: BBa_K404106)) , the beta-globin
-
intron (BBa_K404107),
+
intron
-
the gene of interests (fluorescent proteins mVenus: BBa_I757008 and
+
(BBa_K404107), the gene of interests (fluorescent proteins mVenus:
-
mCherry: BBa_J06504,
+
BBa_I757008 and
-
suicide genes mGMK_TK30: BBa_K404112, mGMK_SR39: BBa_K404315 and CD:
+
mCherry: BBa_J06504, suicide genes mGMK_TK30: BBa_K404112, mGMK_SR39:
-
BBa_K404112)
+
BBa_K404315 and CD: BBa_K404112) and the hGH terminator (BBa_K404108).</p>
-
and the hGH terminator (BBa_K404108).</p>
+
<table class="MsoNormalTable"
-
<table class="MsoTableGrid"
+
  style="border-collapse: collapse;" border="0"
-
  style="border: medium none ; border-collapse: collapse;"
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cellpadding="0" cellspacing="0">
-
border="1" cellpadding="0" cellspacing="0">
+
   <tbody>
   <tbody>
     <tr>
     <tr>
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       <p class="MsoNormal"
       <p class="MsoNormal"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
-
  align="center"><img id="Grafik 1"
+
  align="center"><img
  src="Freiburg10_Modularization_GOI_02_files/image001.gif"
  src="Freiburg10_Modularization_GOI_02_files/image001.gif"
-
  alt="Description: http://partsregistry.org/wiki/images/b/ba/Freiburg10_VectorplasmidBrick_1.png"
+
  alt="Description: Description: http://partsregistry.org/wiki/images/b/ba/Freiburg10_VectorplasmidBrick_1.png"
  height="83" width="102"></p>
  height="83" width="102"></p>
       </td>
       </td>
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  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
  align="center"><span style="font-size: 9pt;"><img
  align="center"><span style="font-size: 9pt;"><img
-
id="Grafik 51"
 
  src="Freiburg10_Modularization_GOI_02_files/image002.gif"
  src="Freiburg10_Modularization_GOI_02_files/image002.gif"
 +
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image002.gif"
  height="69" width="86"></span></p>
  height="69" width="86"></span></p>
       </td>
       </td>
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       <p class="MsoNormal"
       <p class="MsoNormal"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
-
  align="center"><img id="Grafik 6"
+
  align="center"><img
  src="Freiburg10_Modularization_GOI_02_files/image003.gif"
  src="Freiburg10_Modularization_GOI_02_files/image003.gif"
-
  alt="Description: http://partsregistry.org/wiki/images/1/1e/Freiburg10_VectorplasmidBricks_4.png"
+
  alt="Description: Description: http://partsregistry.org/wiki/images/1/1e/Freiburg10_VectorplasmidBricks_4.png"
  height="78" width="116"></p>
  height="78" width="116"></p>
       </td>
       </td>
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       <p class="MsoNormal"
       <p class="MsoNormal"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
-
  align="center"><img id="Grafik 26"
+
  align="center"><img
  src="Freiburg10_Modularization_GOI_02_files/image004.gif"
  src="Freiburg10_Modularization_GOI_02_files/image004.gif"
 +
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image004.gif"
  height="66" width="90"></p>
  height="66" width="90"></p>
       </td>
       </td>
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       <p class="MsoNormal"
       <p class="MsoNormal"
  style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
  style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
-
  align="left"><img id="Grafik 8"
+
  align="left"><img
  src="Freiburg10_Modularization_GOI_02_files/image005.gif"
  src="Freiburg10_Modularization_GOI_02_files/image005.gif"
-
  alt="Description: http://partsregistry.org/wiki/images/0/06/Freiburg10_VectorplasmidBricks_5.png"
+
  alt="Description: Description: http://partsregistry.org/wiki/images/0/06/Freiburg10_VectorplasmidBricks_5.png"
  height="80" width="96"></p>
  height="80" width="96"></p>
       </td>
       </td>
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       <p class="MsoNormal"
       <p class="MsoNormal"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
-
  align="center"><img id="Grafik 4"
+
  align="center"><img
  src="Freiburg10_Modularization_GOI_02_files/image006.gif"
  src="Freiburg10_Modularization_GOI_02_files/image006.gif"
-
  alt="Description: http://partsregistry.org/wiki/images/1/18/Freiburg10_VectorplasmidBricks_2.png"
+
  alt="Description: Description: http://partsregistry.org/wiki/images/1/18/Freiburg10_VectorplasmidBricks_2.png"
  height="79" width="98"></p>
  height="79" width="98"></p>
       </td>
       </td>
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  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
  align="center"><span style="font-size: 9pt;"><img
  align="center"><span style="font-size: 9pt;"><img
-
id="Picture 43"
 
  src="Freiburg10_Modularization_GOI_02_files/image007.jpg"
  src="Freiburg10_Modularization_GOI_02_files/image007.jpg"
 +
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image007.jpg"
  height="80" width="79"></span></p>
  height="80" width="79"></span></p>
       <p class="MsoNormal"
       <p class="MsoNormal"
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  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
  align="center"><span style="font-size: 9pt;"
  align="center"><span style="font-size: 9pt;"
-
  lang="DE">(</span><span
+
  lang="DE">(<span style="color: black;">BBa_K404100</span>)</span></p>
-
style="font-size: 9pt; color: black;" lang="DE">BBa_K404100</span><span
+
-
style="font-size: 9pt;" lang="DE">)</span></p>
+
       </td>
       </td>
       <td
       <td
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  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
  align="center"><span style="font-size: 9pt;"><img
  align="center"><span style="font-size: 9pt;"><img
-
id="Picture 70"
 
  src="Freiburg10_Modularization_GOI_02_files/image008.jpg"
  src="Freiburg10_Modularization_GOI_02_files/image008.jpg"
 +
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image008.jpg"
  height="80" width="80"></span></p>
  height="80" width="80"></span></p>
       <p class="MsoNormal"
       <p class="MsoNormal"
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  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
  align="center"><span style="font-size: 9pt;"><img
  align="center"><span style="font-size: 9pt;"><img
-
id="Picture 61"
 
  src="Freiburg10_Modularization_GOI_02_files/image009.jpg"
  src="Freiburg10_Modularization_GOI_02_files/image009.jpg"
 +
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image009.jpg"
  height="79" width="78"></span></p>
  height="79" width="78"></span></p>
       <p class="MsoNormal"
       <p class="MsoNormal"
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  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
  align="center"><span style="font-size: 9pt;"
  align="center"><span style="font-size: 9pt;"
-
  lang="DE">(</span><span
+
  lang="DE">(<span style="color: black;">BBa_K404107</span>)</span></p>
-
style="font-size: 9pt; color: black;" lang="DE">BBa_K404107</span><span
+
-
style="font-size: 9pt;" lang="DE">)</span></p>
+
       </td>
       </td>
       <td
       <td
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  align="center"><span style="font-size: 9pt;"><img
  align="center"><span style="font-size: 9pt;"><img
-
id="Picture 58"
 
  src="Freiburg10_Modularization_GOI_02_files/image010.jpg"
  src="Freiburg10_Modularization_GOI_02_files/image010.jpg"
 +
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image010.jpg"
  height="79" width="79"></span></p>
  height="79" width="79"></span></p>
       <p class="MsoNormal"
       <p class="MsoNormal"
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  align="center"><span style="font-size: 9pt;"><img
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-
id="Picture 64"
 
  src="Freiburg10_Modularization_GOI_02_files/image011.jpg"
  src="Freiburg10_Modularization_GOI_02_files/image011.jpg"
 +
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image011.jpg"
  height="80" width="80"></span></p>
  height="80" width="80"></span></p>
       <p class="MsoNormal"
       <p class="MsoNormal"
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id="Picture 46"
 
  src="Freiburg10_Modularization_GOI_02_files/image012.jpg"
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 +
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image012.jpg"
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       <p class="MsoNormal"
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id="Picture 67"
 
  src="Freiburg10_Modularization_GOI_02_files/image013.jpg"
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 +
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image013.jpg"
  height="80" width="79"></span></p>
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       <p class="MsoNormal"
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-
id="Picture 109"
 
  src="Freiburg10_Modularization_GOI_02_files/image014.jpg"
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 +
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image014.jpg"
  height="79" width="79"></span></p>
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       <p class="MsoNormal"
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 +
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 +
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-
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 +
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       <p class="MsoNormal"
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  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
  align="center"><span style="font-size: 9pt;"><img
  align="center"><span style="font-size: 9pt;"><img
-
id="Grafik 2057"
 
  src="Freiburg10_Modularization_GOI_02_files/image018.gif"
  src="Freiburg10_Modularization_GOI_02_files/image018.gif"
 +
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image018.gif"
  height="116" width="555"></span></p>
  height="116" width="555"></span></p>
       </td>
       </td>
Line 527: Line 560:
   </tbody>
   </tbody>
</table>
</table>
-
<h3>Cloning and Combination Strategies for the Vectorplasmid </h3>
+
<h3><a name="_Toc275794995">Cloning and Combination
 +
Strategies for the
 +
Vectorplasmid</a> </h3>
<p class="MsoNormal">Organization of the recombinant viral
<p class="MsoNormal">Organization of the recombinant viral
-
DNA was modified
+
DNA was modified ensuring
-
ensuring several layers of specificity to our systems including a
+
several layers of specificity to our systems including a tumor-specific
-
tumor-specific promoter and suicide genes encoding prodrug convertases.
+
promoter and suicide genes encoding prodrug convertases. In order to
-
In
+
modularize
-
order to modularize the rAAV sequence, each plasmid element (Figure 1)
+
the rAAV sequence, each plasmid element (Figure 1) was PCR-amplified
-
was PCR-amplified and cloned into the iGEM standard plasmid pSB1C3.
+
and cloned
-
Furthermore, the iGEM
+
into the iGEM standard plasmid pSB1C3. Furthermore, the iGEM team
-
team Freiburg_Bioware 2010 performed three site-directed mutagenesis in
+
Freiburg_Bioware 2010 performed three site-directed mutagenesis in the
-
the
+
gene of
-
gene of interest TK30 (BBa_K404109) and cytosine deaminase (<span
+
interest TK30 (BBa_K404109) and cytosine deaminase (<span
  style="font-size: 9pt; line-height: 200%;">BBa_K404112</span>)
  style="font-size: 9pt; line-height: 200%;">BBa_K404112</span>)
-
for deletion of
+
for deletion of PstI and NgoMIV
-
PstI and NgoMIV iGEM site (for further information see the results page
+
iGEM site (for further information see the results page of ‘Arming –
-
of
+
Killing
-
‘Arming – Killing the tumor’). Since the inverted terminal repeats
+
the tumor’). Since the inverted terminal repeats (ITRs) are GC-rich
-
(ITRs) are
+
regions
-
GC-rich regions forming T-shaped hairpins during replication, PCR
+
forming T-shaped hairpins during replication, PCR amplification was not
-
amplification
+
possible. Hence a cloning strategy was developed by the iGEM team
-
was not possible. Hence a cloning strategy was developed by the iGEM
+
Freiburg in
-
team
+
order to provide BioBrick-compatible ITRs (see ).</p>
-
Freiburg in order to provide BioBrick-compatible ITRs (see ).</p>
+
<p class="MsoNormal">In Figure 2 the schematic overview of
<p class="MsoNormal">In Figure 2 the schematic overview of
the modularization
the modularization
Line 556: Line 590:
required for functional vectorplasmids.</p>
required for functional vectorplasmids.</p>
<div align="center">
<div align="center">
-
<table class="MsoTableGrid"
+
<table class="MsoNormalTable"
-
  style="border: medium none ; border-collapse: collapse;"
+
  style="border-collapse: collapse;" border="0"
-
border="1" cellpadding="0" cellspacing="0">
+
cellpadding="0" cellspacing="0">
   <tbody>
   <tbody>
     <tr style="height: 26.15pt;">
     <tr style="height: 26.15pt;">
Line 566: Line 600:
       <p class="MsoNormal"
       <p class="MsoNormal"
  style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
  style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
-
id="Grafik 2"
 
  src="Freiburg10_Modularization_GOI_02_files/image019.gif"
  src="Freiburg10_Modularization_GOI_02_files/image019.gif"
-
  alt="Description: http://partsregistry.org/wiki/images/1/1c/Freiburg10_Vectorplasmid_cloning.png"
+
  alt="Description: Description: http://partsregistry.org/wiki/images/1/1c/Freiburg10_Vectorplasmid_cloning.png"
  height="272" width="439"></p>
  height="272" width="439"></p>
       <p class="MsoCaption" style="text-indent: 0cm;"><a
       <p class="MsoCaption" style="text-indent: 0cm;"><a
Line 599: Line 632:
leftITR_CMV together can be observed. </p>
leftITR_CMV together can be observed. </p>
<div align="center">
<div align="center">
-
<table class="MsoTableGrid"
+
<table class="MsoNormalTable"
-
  style="border: medium none ; border-collapse: collapse;"
+
  style="border-collapse: collapse;" border="0"
-
border="1" cellpadding="0" cellspacing="0">
+
cellpadding="0" cellspacing="0">
   <tbody>
   <tbody>
     <tr style="height: 23.95pt;">
     <tr style="height: 23.95pt;">
Line 609: Line 642:
       <p class="MsoNormal"
       <p class="MsoNormal"
  style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
  style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
-
id="Grafik 74"
 
  src="Freiburg10_Modularization_GOI_02_files/image020.gif"
  src="Freiburg10_Modularization_GOI_02_files/image020.gif"
 +
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image020.gif"
  height="530" width="452"></p>
  height="530" width="452"></p>
       <p class="MsoCaption" style="text-indent: 0cm;"><a
       <p class="MsoCaption" style="text-indent: 0cm;"><a
Line 637: Line 670:
strategy.</p>
strategy.</p>
<div align="center">
<div align="center">
-
<table class="MsoTableGrid"
+
<table class="MsoNormalTable"
-
  style="border: medium none ; border-collapse: collapse;"
+
  style="border-collapse: collapse;" border="0"
-
border="1" cellpadding="0" cellspacing="0">
+
cellpadding="0" cellspacing="0">
   <tbody>
   <tbody>
     <tr style="height: 129.95pt;">
     <tr style="height: 129.95pt;">
Line 649: Line 682:
       <p class="MsoNormal"
       <p class="MsoNormal"
  style="margin-bottom: 0.0001pt; text-align: right; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
  style="margin-bottom: 0.0001pt; text-align: right; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
-
  align="right"><img id="Grafik 77"
+
  align="right"><img
  src="Freiburg10_Modularization_GOI_02_files/image021.gif"
  src="Freiburg10_Modularization_GOI_02_files/image021.gif"
-
  alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\09.09_Cloning_leftITR_beta to pCMV and phTERT.png"
+
  alt="Description: Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\09.09_Cloning_leftITR_beta to pCMV and phTERT.png"
  height="194" width="397"></p>
  height="194" width="397"></p>
       <p class="MsoCaption"
       <p class="MsoCaption"
Line 680: Line 713:
After
After
ligation has been carried out, <i>E. coli</i> XL-1B cells
ligation has been carried out, <i>E. coli</i> XL-1B cells
-
were transformed and
+
were transformed and incubated
-
incubated over night at 37°C. Picking clones from the transformation
+
over night at 37°C. Picking clones from the transformation plate was
-
plate was
+
performed
-
performed the following day and DYT medium was inoculated incubating
+
the following day and DYT medium was inoculated incubating overnight.
-
overnight.
+
Plasmid
-
Plasmid DNA was isolated and test digestion revealed that cloning was
+
DNA was isolated and test digestion revealed that cloning was
-
successful obtaining the composite part leftITR_CMV_<i>beta-globin</i>
+
successful
-
intron (BBa_K404117).</p>
+
obtaining the composite part leftITR_CMV_<i>beta-globin</i>
 +
intron
 +
(BBa_K404117).</p>
<p class="MsoNormal">Plasmid production incorporating all
<p class="MsoNormal">Plasmid production incorporating all
required elements for
required elements for
Line 693: Line 728:
was
was
performed by fusing the downstream elements consisting of the hGH
performed by fusing the downstream elements consisting of the hGH
-
terminator and
+
terminator
-
right ITR to the intermediate part providing the gene of interest and
+
and right ITR to the intermediate part providing the gene of interest
-
the
+
and the
promoter fused to the left ITR. Figure 5 demonstrates the assembly
promoter fused to the left ITR. Figure 5 demonstrates the assembly
performed
performed
Line 707: Line 742:
obtaining
obtaining
the vectorplasmid pSB1C3_leftITR_phTERT_<i>beta-globin</i>
the vectorplasmid pSB1C3_leftITR_phTERT_<i>beta-globin</i>
-
intron_mVenus_hGH_rightITR
+
intron_mVenus_hGH_rightITR (<span style="color: black;">BBa_K404124</span>).
-
(<span style="line-height: 200%; color: black;">BBa_K404124</span>).
+
</p>
</p>
<div align="center">
<div align="center">
-
<table class="MsoTableGrid"
+
<table class="MsoNormalTable"
-
  style="border: medium none ; border-collapse: collapse;"
+
  style="border-collapse: collapse;" border="0"
-
border="1" cellpadding="0" cellspacing="0">
+
cellpadding="0" cellspacing="0">
   <tbody>
   <tbody>
     <tr style="height: 136.9pt;">
     <tr style="height: 136.9pt;">
Line 721: Line 755:
       <p class="MsoNormal"
       <p class="MsoNormal"
  style="margin-bottom: 0.0001pt; text-align: right; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
  style="margin-bottom: 0.0001pt; text-align: right; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
-
  align="right"><img id="Grafik 80"
+
  align="right"><img
  src="Freiburg10_Modularization_GOI_02_files/image022.gif"
  src="Freiburg10_Modularization_GOI_02_files/image022.gif"
-
  alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\18.09_Cloning_Full_phTERT_mVenus.png"
+
  alt="Description: Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\18.09_Cloning_Full_phTERT_mVenus.png"
  height="184" width="397"></p>
  height="184" width="397"></p>
       <p class="MsoCaption" style="text-indent: 0cm;"><a
       <p class="MsoCaption" style="text-indent: 0cm;"><a
Line 754: Line 788:
with
with
helper plasmids required for capsid assembly&nbsp; and genome
helper plasmids required for capsid assembly&nbsp; and genome
-
encapsidation (pRC and
+
encapsidation
-
pHelper) in a molar ratio of 1:1:1 (pGOI:pRC:pHelper). Virus particles
+
(pRC and pHelper) in a molar ratio of 1:1:1 (pGOI:pRC:pHelper). Virus
 +
particles
containing the single stranded DNA were harvested 72-hours post
containing the single stranded DNA were harvested 72-hours post
transfection
transfection
Line 769: Line 804:
flow
flow
cytometry analysis.</p>
cytometry analysis.</p>
-
<h3>Testing functionality of Assembled Vectorplasmid</h3>
+
<h3><a name="_Toc275794996">Testing functionality of
 +
Assembled Vectorplasmid</a></h3>
<h4 style="margin-left: 0cm; text-indent: 0cm;"><span
<h4 style="margin-left: 0cm; text-indent: 0cm;"><span
  lang="DE">Fluorescence</span>
  lang="DE">Fluorescence</span>
Line 778: Line 814:
HT1080
HT1080
cells and non-transduced cells could be easily distinguished. In Figure
cells and non-transduced cells could be easily distinguished. In Figure
-
6 cells were excited with 505nm and fluorescence emission at 536nm was
+
6 cells
-
detected. Therefore, successful infection of tumor cells by recombinant
+
were excited with 505nm and fluorescence emission at 536nm was
-
viral
+
detected.
-
particles carrying the assembled vectorplasmid coding for mVenus could
+
Therefore, successful infection of tumor cells by recombinant viral
-
be
+
particles
 +
carrying the assembled vectorplasmid coding for mVenus could be
demonstrated. </p>
demonstrated. </p>
-
<table class="MsoTableGrid"
+
<table class="MsoNormalTable"
-
  style="border: medium none ; border-collapse: collapse;"
+
  style="border-collapse: collapse;" border="0"
-
border="1" cellpadding="0" cellspacing="0">
+
cellpadding="0" cellspacing="0">
   <tbody>
   <tbody>
     <tr>
     <tr>
Line 796: Line 833:
       <p class="MsoNormal"
       <p class="MsoNormal"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
-
  align="center"><img id="Grafik 18"
+
  align="center"><img
  src="Freiburg10_Modularization_GOI_02_files/image023.jpg"
  src="Freiburg10_Modularization_GOI_02_files/image023.jpg"
-
  alt="Description: Freiburg10_2Transd30µg_unverd_2_(c1).JPG (1388×1040)"
+
  alt="Description: Description: Freiburg10_2Transd30µg_unverd_2_(c1).JPG (1388×1040)"
  height="218" width="264"></p>
  height="218" width="264"></p>
       </td>
       </td>
Line 808: Line 845:
       <p class="MsoNormal"
       <p class="MsoNormal"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
-
  align="center"><img id="Grafik 19"
+
  align="center"><img
  src="Freiburg10_Modularization_GOI_02_files/image024.jpg"
  src="Freiburg10_Modularization_GOI_02_files/image024.jpg"
-
  alt="Description: http://2010.igem.org/wiki/images/f/f1/Freiburg10_2Transd30%C2%B5g_unverd_%28c1%29.JPG"
+
  alt="Description: Description: http://2010.igem.org/wiki/images/f/f1/Freiburg10_2Transd30%C2%B5g_unverd_%28c1%29.JPG"
  height="220" width="242"></p>
  height="220" width="242"></p>
       </td>
       </td>
Line 822: Line 859:
       <p class="MsoNormal"
       <p class="MsoNormal"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
-
  align="center"><img id="Grafik 16"
+
  align="center"><img
  src="Freiburg10_Modularization_GOI_02_files/image025.jpg"
  src="Freiburg10_Modularization_GOI_02_files/image025.jpg"
-
  alt="Description: http://2010.igem.org/wiki/images/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"
+
  alt="Description: Description: http://2010.igem.org/wiki/images/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"
  height="195" width="258"></p>
  height="195" width="258"></p>
       </td>
       </td>
Line 834: Line 871:
       <p class="MsoNormal"
       <p class="MsoNormal"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
-
  align="center"><img id="Grafik 17"
+
  align="center"><img
  src="Freiburg10_Modularization_GOI_02_files/image026.jpg"
  src="Freiburg10_Modularization_GOI_02_files/image026.jpg"
-
  alt="Description: http://2010.igem.org/wiki/images/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"
+
  alt="Description: Description: http://2010.igem.org/wiki/images/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"
  height="194" width="257"></p>
  height="194" width="257"></p>
       </td>
       </td>
Line 856: Line 893:
<p class="MsoNormal">&nbsp;</p>
<p class="MsoNormal">&nbsp;</p>
<h4 style="margin-left: 0cm; text-indent: 0cm;"><span
<h4 style="margin-left: 0cm; text-indent: 0cm;"><span
-
  lang="DE">Analysis</span><span lang="DE">
+
  lang="DE">Analysis </span>of
-
</span>of Target Cells by Flow Cytometry demonstrates GOI
+
Target Cells by Flow Cytometry demonstrates GOI Expression</h4>
-
Expression</h4>
+
<p class="MsoNormal">Characterizing the function of the
<p class="MsoNormal">Characterizing the function of the
hGH terminator, the <i>beta-globin</i>
hGH terminator, the <i>beta-globin</i>
intron and the complete plasmid, several approaches were conducted
intron and the complete plasmid, several approaches were conducted
-
followed by
+
followed by analysis
-
analysis via flow cytometry. </p>
+
via flow cytometry. </p>
<h5>Influence of hGH terminator BioBrick on GOI Expression</h5>
<h5>Influence of hGH terminator BioBrick on GOI Expression</h5>
<p class="MsoNormal">The iGEM team Freiburg provides the
<p class="MsoNormal">The iGEM team Freiburg provides the
Line 870: Line 906:
almost every
almost every
eukaryotic mRNA is processed at their 3´ and 5´end except for histone
eukaryotic mRNA is processed at their 3´ and 5´end except for histone
-
mRNAs (Millevoi et al. 2006). Pre-mRNAs contain two canonical conserved
+
mRNAs
-
sequences. First, the
+
(Millevoi et al. 2006). Pre-mRNAs contain two canonical conserved
-
polyadenylation signal “AATAAA” which is recognized by the multiprotein
+
sequences.
-
complex
+
First, the polyadenylation signal “AATAAA” which is recognized by the
-
and second the GT-rich region (downstream sequence element, DSE) which
+
multiprotein complex and second the GT-rich region (downstream sequence
-
is
+
element, DSE) which is located 30 nucleotides downstream of the
-
located 30 nucleotides downstream of the cleavage site. The assembled
+
cleavage site.
-
3´end-processing machinery cleaves the mRNA transcript immediately
+
The assembled 3´end-processing machinery cleaves the mRNA transcript
-
after a
+
immediately after a CA-nucleotide therefore defining the cleavage site
-
CA-nucleotide therefore defining the cleavage site (Danckwardt et al.
+
(Danckwardt et al. 2008)<span
-
2008)<span style="font-size: 12pt; line-height: 200%;">. </span>Recombinant
+
style="font-size: 12pt; line-height: 200%;">. </span>Recombinant
vectorplasmids were engineered containing the inverted terminal repeats
vectorplasmids were engineered containing the inverted terminal repeats
(ITRs),
(ITRs),
Line 893: Line 929:
hGH
hGH
termination signal is significantly reduced as shown in Figure 7 and
termination signal is significantly reduced as shown in Figure 7 and
-
Figure 8 confirming the expected results that hGH is essential for mRNA
+
Figure 8
-
processing.
+
confirming the expected results that hGH is essential for mRNA
-
The iGEM team Freiburg_Bioware 2010 therefore suggests using the
+
processing. The
-
provided hGH
+
iGEM team Freiburg_Bioware 2010 therefore suggests using the provided
 +
hGH
termination signal within the Virus Construction Kit for optimal gene
termination signal within the Virus Construction Kit for optimal gene
expression.</p>
expression.</p>
-
<table class="MsoTableGrid"
+
<table class="MsoNormalTable"
-
  style="border: medium none ; border-collapse: collapse;"
+
  style="border-collapse: collapse;" border="0"
-
border="1" cellpadding="0" cellspacing="0">
+
cellpadding="0" cellspacing="0">
   <tbody>
   <tbody>
     <tr>
     <tr>
Line 912: Line 949:
       <p class="MsoNormal"
       <p class="MsoNormal"
  style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img
  style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img
-
id="Grafik 30"
 
  src="Freiburg10_Modularization_GOI_02_files/image027.gif"
  src="Freiburg10_Modularization_GOI_02_files/image027.gif"
 +
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image027.gif"
  height="408" width="629"></p>
  height="408" width="629"></p>
       </td>
       </td>
Line 926: Line 963:
       <p class="MsoNormal"
       <p class="MsoNormal"
  style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
  style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
-
id="Grafik 2049"
 
  src="Freiburg10_Modularization_GOI_02_files/image028.gif"
  src="Freiburg10_Modularization_GOI_02_files/image028.gif"
 +
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image028.gif"
  height="410" width="636"></p>
  height="410" width="636"></p>
       </td>
       </td>
Line 937: Line 974:
       <p class="MsoNormal"
       <p class="MsoNormal"
  style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><a
  style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><a
-
  name="_Ref275784539"><b>Figure </b></a><b>7</b><b>:</b>
+
  name="_Ref275784539"><b>Figure </b></a><b>7:</b>
       <b>Flow cytometry analysis of vectorplasmids with and
       <b>Flow cytometry analysis of vectorplasmids with and
without hGH terminator.</b> </p>
without hGH terminator.</b> </p>
Line 969: Line 1,006:
</table>
</table>
<p class="MsoNormal"><span lang="DE">&nbsp;</span></p>
<p class="MsoNormal"><span lang="DE">&nbsp;</span></p>
-
<table class="MsoTableGrid"
+
<table class="MsoNormalTable"
-
  style="border: medium none ; border-collapse: collapse;"
+
  style="border-collapse: collapse;" border="0"
-
border="1" cellpadding="0" cellspacing="0">
+
cellpadding="0" cellspacing="0">
   <tbody>
   <tbody>
     <tr>
     <tr>
Line 981: Line 1,018:
       <p class="MsoNormal"
       <p class="MsoNormal"
  style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
  style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-
       <img id="Diagramm 3"
+
       <img src="Freiburg10_Modularization_GOI_02_files/image029.gif"
-
  src="Freiburg10_Modularization_GOI_02_files/image029.gif"
+
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  height="355" width="473"></p>
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       <p class="MsoCaption" style="text-indent: 0cm;"><a
       <p class="MsoCaption" style="text-indent: 0cm;"><a
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intron BioBrick is assumed to
intron BioBrick is assumed to
enhance eukaryotic gene expression (Nott et al. 2003). Analysis was
enhance eukaryotic gene expression (Nott et al. 2003). Analysis was
-
conducted as described for the hGH terminator experiment
+
conducted
-
(see above). As shown in Figure 9 and Figure 10 the vectorplasmid
+
as described for the hGH terminator experiment (see above). As shown in
-
missing the <i>beta-globin</i>
+
Figure
-
intron showed a negligible difference in mVenus expression compared to
+
9 and Figure 10 the vectorplasmid missing the <i>beta-globin</i>
-
viral
+
intron showed
-
genomes containing the <i>beta-globin</i> intron.
+
a negligible difference in mVenus expression compared to viral genomes
-
Considering these results and
+
containing the <i>beta-globin</i> intron. Considering
-
taking into account that a constant volume of viral particles has been
+
these results and taking
-
used for
+
into account that a constant volume of viral particles has been used
 +
for
transduction, the difference between the construct containing and
transduction, the difference between the construct containing and
lacking the
lacking the
beta-globin intron is minimal. Since packaging efficiency of the AAV-2
beta-globin intron is minimal. Since packaging efficiency of the AAV-2
decreases with increasing sizes of the insert (Dong et al. 1996), the
decreases with increasing sizes of the insert (Dong et al. 1996), the
-
iGEM team Freiburg_Bioware suggests using the <i>beta-globin </i>intron
+
iGEM team
-
in dependence on the size of your transgene.</p>
+
Freiburg_Bioware suggests using the <i>beta-globin </i>intron
-
<table class="MsoTableGrid"
+
in dependence on
-
  style="border: medium none ; width: 490.75pt; border-collapse: collapse;"
+
the size of your transgene.</p>
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id="Grafik 55"
 
  src="Freiburg10_Modularization_GOI_02_files/image030.gif"
  src="Freiburg10_Modularization_GOI_02_files/image030.gif"
 +
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image030.gif"
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id="Grafik 63"
 
  src="Freiburg10_Modularization_GOI_02_files/image031.gif"
  src="Freiburg10_Modularization_GOI_02_files/image031.gif"
 +
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image031.gif"
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</table>
</table>
<p class="MsoNormal"><span lang="DE">&nbsp;</span></p>
<p class="MsoNormal"><span lang="DE">&nbsp;</span></p>
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<table class="MsoTableGrid"
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<table class="MsoNormalTable"
-
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  style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
-
  align="center"><img id="Diagramm 57"
+
  align="center"><img
  src="Freiburg10_Modularization_GOI_02_files/image032.gif"
  src="Freiburg10_Modularization_GOI_02_files/image032.gif"
 +
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image032.gif"
  height="332" width="450"></p>
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<p class="MsoNormal" style="text-indent: 0cm;">&nbsp;</p>
<p class="MsoNormal" style="text-indent: 0cm;">&nbsp;</p>
<h5>Functionality of the Full Assembled Vectorplasmid
<h5>Functionality of the Full Assembled Vectorplasmid
-
Demonstrated by GOI Expression
+
Demonstrated by GOI
-
</h5>
+
Expression </h5>
<p class="MsoNormal">After assembly of plasmids containing
<p class="MsoNormal">After assembly of plasmids containing
all required elements
all required elements
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stably
stably
expressing E1A and E1B proteins were transfected with three plasmids
expressing E1A and E1B proteins were transfected with three plasmids
-
&nbsp;(pHelper,
+
&nbsp;(pHelper, pRC, pGOI). Virus particles were harvested 72-hours
-
pRC, pGOI). Virus particles were harvested 72-hours post-transfection
+
post-transfection and the tumor cell line HT1080 was transduced with
-
and the
+
the
-
tumor cell line HT1080 was transduced with the recombinant viral
+
recombinant viral vectors encapsidating the gene of interest mVenus
-
vectors
+
(BBa_I757008).</p>
-
encapsidating the gene of interest mVenus (BBa_I757008).</p>
+
<p class="MsoNormal">The iGEM team Freiburg_Bioware 2010
<p class="MsoNormal">The iGEM team Freiburg_Bioware 2010
-
compared the standard-plasmid
+
compared the
-
containing a subcloned mVenus (pAAV_mVenus, derived from the Stratagene
+
standard-plasmid containing a subcloned mVenus (pAAV_mVenus, derived
-
system)
+
from the
-
with the assembled plasmid pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
+
Stratagene system) with the assembled plasmid
-
(pSB1C3_mVenus: BBa_K404119). Fluorescence expression data obtained by
+
pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (pSB1C3_mVenus:
-
flow
+
BBa_K404119).
-
cytometry analysis are shown in Figure 11 and Figure 12. Comparing
+
Fluorescence expression data obtained by flow cytometry analysis are
-
mVenus expression
+
shown in
-
of the standard plasmid and the modified, assembled plasmid reveals
+
Figure 11 and Figure 12. Comparing mVenus expression of the standard
-
that
+
plasmid
-
biological functionality of the reassembled plasmid was confirmed. </p>
+
and the modified, assembled plasmid reveals that biological
-
<table class="MsoTableGrid"
+
functionality of
-
  style="border: medium none ; border-collapse: collapse;"
+
the reassembled plasmid was confirmed. </p>
-
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+
<table class="MsoNormalTable"
 +
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     <tr>
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-
id="Grafik 2068"
 
  src="Freiburg10_Modularization_GOI_02_files/image033.gif"
  src="Freiburg10_Modularization_GOI_02_files/image033.gif"
 +
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image033.gif"
  height="409" width="634"></p>
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id="Grafik 89"
 
  src="Freiburg10_Modularization_GOI_02_files/image034.gif"
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 +
alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image034.gif"
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<p class="MsoNormal"><span lang="DE">&nbsp;</span></p>
<p class="MsoNormal"><span lang="DE">&nbsp;</span></p>
<p class="MsoNormal"><span lang="DE">&nbsp;</span></p>
<p class="MsoNormal"><span lang="DE">&nbsp;</span></p>
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<table class="MsoTableGrid"
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<table class="MsoNormalTable"
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-
  align="center"><img id="Diagramm 90"
+
  align="center"><img
  src="Freiburg10_Modularization_GOI_02_files/image035.gif"
  src="Freiburg10_Modularization_GOI_02_files/image035.gif"
 +
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  lang="DE">Dong, J.Y., Fan,
  lang="DE">Dong, J.Y., Fan,
P.D. &amp; Frizzell, R.A., 1996. Quantitative analysis of the
P.D. &amp; Frizzell, R.A., 1996. Quantitative analysis of the
-
packaging
+
packaging capacity
-
capacity of recombinant adeno-associated virus. <i>Human gene
+
of recombinant adeno-associated virus. <i>Human gene therapy</i>,
-
therapy</i>,
+
7(17),
-
7(17), pp.2101-12. Available at:
+
pp.2101-12. Available at: http://www.ncbi.nlm.nih.gov/pubmed/8934224.</span></p>
-
http://www.ncbi.nlm.nih.gov/pubmed/8934224.</span></p>
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<p style="margin-left: 24pt; text-indent: -24pt;"><span
<p style="margin-left: 24pt; text-indent: -24pt;"><span
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Available at: http://www.ncbi.nlm.nih.gov/pubmed/12702819.</span></p>
Available at: http://www.ncbi.nlm.nih.gov/pubmed/12702819.</span></p>
<p style="margin-left: 24pt; text-indent: -24pt;">&nbsp;</p>
<p style="margin-left: 24pt; text-indent: -24pt;">&nbsp;</p>
 +
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Revision as of 16:39, 25 October 2010

 

Contents

Modularization GOI/Vector Plasmid. 1

Introduction to Modularization. 1

Recombinant and Modular Vector Plasmid Carrying GOI 1

Cloning and Combination Strategies for the Vectorplasmid. 2

Testing functionality of Assembled Vectorplasmid. 3

 

 

 

Modularization GOI/Vector Plasmid

Introduction to Modularization

Producing recombinant virus particles for therapeutical means is, besides specifically target cells, purification and quantification assays of AAV-2, one intention of the Virus Construction Kit provided by the iGEM team Freiburg_Bioware 2010. For obtaining a modular toolkit, the complex components of AAV-2 were extracted and redesigned to match the iGEM standard. Functional activity was tested in cell culture.

Differing from the wildtype AAV-2 genome, the Helper Free System provided by Stratagene comprises three plasmids and a specialized production cell line. AAV-293 cells derived from the HEK cell line express the stably integrated E1A and E1B helper proteins for efficient virus production. The plasmid containing the inverted terminal repeats (ITRs) is encapsidated into the preformed capsids after production of single-stranded DNA therefore also known as vectorplasmid (pGOI). Promoter, beta-globin intron and the hGH terminator signal are flanked by the ITRs and serve in the host cell for regulation of transgene expression. In addition to that, the plasmid coding for the Rep and Cap proteins (pRC) can be provided in trans leading to a layer of specificity due to the fact that the two genes are not packaged into the capsid since lacking of the ITRs impairs encapsidation. Another advantage of the Helper Free System can be attributed to cotransfection of another helper plasmid (pHelper), which provides the necessary proteins normally obtained by superinfection with helper viruses such as adenovirus or herpes simplex virus. These helper genes are required for full viral assembly by regulating gene expression of Rep and Cap proteins.

Recombinant and Modular Vector Plasmid Carrying GOI

The iGEM team Freiburg_Bioware 2010 provides a modular Virus Construction Kit for therapeutical applications, quantification assays and purification approaches depending on capsid modifications and the gene of interest flanked by the inverted terminal repeats (ITRs. In order to produce BioBrick-compatible standardized biological parts, we reengineered the plasmids and added new components for gene therapy approaches and analysis of biological activity of assembled BioBrick parts. Each element required for intact and functional plasmids comprising the ITRs, a promoter, a putative enhancer element and the hGH terminator was PCR amplified and fused together de novo. As shown in Figure 1, the vectorplasmid was assembled with the produced BioBricks consisting of the left and right ITR (BBa_K404100 and BBa_K404101), a promoter (pCMV :BBa_K404102 or phTERT: BBa_K404106)) , the beta-globin intron (BBa_K404107), the gene of interests (fluorescent proteins mVenus: BBa_I757008 and mCherry: BBa_J06504, suicide genes mGMK_TK30: BBa_K404112, mGMK_SR39: BBa_K404315 and CD: BBa_K404112) and the hGH terminator (BBa_K404108).

Description: Description: http://partsregistry.org/wiki/images/b/ba/Freiburg10_VectorplasmidBrick_1.png

Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image002.gif

Description: Description: http://partsregistry.org/wiki/images/1/1e/Freiburg10_VectorplasmidBricks_4.png

Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image004.gif

Description: Description: http://partsregistry.org/wiki/images/0/06/Freiburg10_VectorplasmidBricks_5.png

Description: Description: http://partsregistry.org/wiki/images/1/18/Freiburg10_VectorplasmidBricks_2.png

Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image007.jpg

Left ITR

(BBa_K404100)

Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image008.jpg

pCMV

(BBa_K404102)

Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image009.jpg

Beta-globin intron

(BBa_K404107)

Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image010.jpg

mVenus (BBa_I757008)

Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image011.jpg

hGH terminator

(BBa_K404108)

Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image012.jpg

Right ITR

(BBa_K404101)

 

Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image013.jpg

phTERT (BBa_K404106)

 

Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image014.jpg

mCherry

(BBa_J06504)

 

 

 

 

 

Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image015.jpg

Cytosine deaminase (BBa_K404112)

 

 

 

 

 

 

Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image016.jpg

mGMK_SR39

(BBa_K404315)

 

 

 

 

 

Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image017.jpg

mGMK_TK30

(BBa_K404113)

 

 

Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image018.gif

Figure 1: Overview of the theoretical sequence of each BioBrick provided within the Virus Construction Kit for an intact and fully functional rAAV genome. The plasmid in the lowest panel was used for tumor killing in combination with plasmids coding for modified capsid proteins. More detailed infomartion about these constructs can be found under ‘Arming: Killing the tumor’ and ‘N-terminal fusion for Targeting’.

Cloning and Combination Strategies for the Vectorplasmid

Organization of the recombinant viral DNA was modified ensuring several layers of specificity to our systems including a tumor-specific promoter and suicide genes encoding prodrug convertases. In order to modularize the rAAV sequence, each plasmid element (Figure 1) was PCR-amplified and cloned into the iGEM standard plasmid pSB1C3. Furthermore, the iGEM team Freiburg_Bioware 2010 performed three site-directed mutagenesis in the gene of interest TK30 (BBa_K404109) and cytosine deaminase (BBa_K404112) for deletion of PstI and NgoMIV iGEM site (for further information see the results page of ‘Arming – Killing the tumor’). Since the inverted terminal repeats (ITRs) are GC-rich regions forming T-shaped hairpins during replication, PCR amplification was not possible. Hence a cloning strategy was developed by the iGEM team Freiburg in order to provide BioBrick-compatible ITRs (see ).

In Figure 2 the schematic overview of the modularization process can be seen which has been followed to conduct the assembly steps required for functional vectorplasmids.

Description: Description: http://partsregistry.org/wiki/images/1/1c/Freiburg10_Vectorplasmid_cloning.png

Figure 2: Assembly procedure for fusion of BioBricks and composite parts to a fully assembled and functional plasmid coding for your gene of interest. This plasmid can be cotransfected with two helper plasmids providing protein for assembly and encapsidating of the rAAV genome (your gene of interest) into the capsids.

 

The iGEM team Freiburg_Bioware provides two examples demonstrating the assembly procedure for constructing vectorplasmids. The first representative example is the fusion of the BioBrick part beta-globin to the composite parts containing the 5´ elements of the plasmids, which are left ITR and CMV or phTERT promoter, respectively.

As shown in Figure 3 the theoretical cloning performed for assembling the BioBricks beta-globin intron and leftITR_CMV together can be observed.

Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image020.gif

Figure 3: Theoretical cloning of the composite part leftITR_CMV to the beta-globin intron BioBrick leading to the plasmid leftITR_CMV_beta-globin intron.

 

The plasmids were digested with both XbaI and PstI (beta-globin intron: BBa_K404107) or SpeI and PstI (leftITR_CMV) and loaded on an agarose gel. As demonstrated in the preparative gel in Figure 4, the expected bands could be detected under UV light and the extracted DNA could be successfully ligated. Each assembly step for producing BioBrick intermediates was conducted following the same strategy.

 

Description: Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\09.09_Cloning_leftITR_beta to pCMV and phTERT.png

Figure 4: Assembly intermediate in fusion of the vectorplasmids containing different promoters. Fusion of the BioBrick part beta-globin (BBa_K404107) intron to the composite parts leftITR_pCMV and leftITR_phTERT, respectively, was performed following the BioBrick assembly strategy by digesting the insert with PstI and XbaI and the vectors with SpeI and PstI. The left lane shows the expected fragment at around 560 bp which corresponds to the beta-globin intron fragment, in contrast to the two lanes in the center and on the right which correspond to linearized plasmids after digesting with above mentioned iGEM restriction sites. M, GeneRuler DNA ladder mix; Insert, pSB1C3_beta-globin intron; Vector pCMV, pSB1C3_leftITR_pCMV; Vector phTERT, pSB1C3_leftITR_phTERT.

 

Separated fragments were extracted using the Gel Extraction Kit provided by Qiagen (Hilden, Germany) and ligated with T4-ligase. After ligation has been carried out, E. coli XL-1B cells were transformed and incubated over night at 37°C. Picking clones from the transformation plate was performed the following day and DYT medium was inoculated incubating overnight. Plasmid DNA was isolated and test digestion revealed that cloning was successful obtaining the composite part leftITR_CMV_beta-globin intron (BBa_K404117).

Plasmid production incorporating all required elements for transgene expression and genome encapsidation into empty viral capsids was performed by fusing the downstream elements consisting of the hGH terminator and right ITR to the intermediate part providing the gene of interest and the promoter fused to the left ITR. Figure 5 demonstrates the assembly performed with pSB1C3_leftITR_phTERT_beta-globin intron_mVenus and pSB1C3_hGH_rightITR (BBa_K404116). The fragment obtained after digestion on the left lane fits to the hGH-terminator_rightITR length. The isolated fragments were ligated and successful assembly was confirmed by test digestion obtaining the vectorplasmid pSB1C3_leftITR_phTERT_beta-globin intron_mVenus_hGH_rightITR (BBa_K404124).

Description: Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\18.09_Cloning_Full_phTERT_mVenus.png

Figure 5: Modularization of the assembled vectorplasmid containing the phTERT promoter and mVenus as gene of interest. Fusion of the composite pSB1C3_leftITR_phTERT_beta-globin intron_mVenus part  to the composite parts pSB1C3_hGH_rightITR was performed following the BioBrick assembly strategy by digesting the insert with XbaI and PstI and the vector with SpeI and PstI. The left lane corresponds to linearized plasmid after digesting with above mentioned iGEM restriction sites whereas the right lane reveals an intensive band at around 650 bp confirming the expected size of 657 bp of hGH_rITR. M, GeneRuler DNA ladder mix; Vector, pSB1C3_leftITR_phTERT_beta-globin intron_mVenus; Insert, pSB1C3_ pSB1C3_hGH_rightITR.

 

Since cloning does not confirm biological activity, we analyzed the plasmids and their functional components, hGH terminator and beta-globin intron, in cell culture. Assembled plasmids have been cotransfected, using AAV-293 cells, which provide the stable integrated E1A and E1B genes, with helper plasmids required for capsid assembly  and genome encapsidation (pRC and pHelper) in a molar ratio of 1:1:1 (pGOI:pRC:pHelper). Virus particles containing the single stranded DNA were harvested 72-hours post transfection and HT1080 cells transduced with constant volumes of viral vectors. 48-hours post infection; transduced cells expressing the gene of interest were analyzed by flow cytometry. Facilitating and demonstrating the analysis of functionality of the assembled plasmid, mVenus was used in first place since fluorescent proteins enable facile visualization using fluorescent microscopy and flow cytometry analysis.

Testing functionality of Assembled Vectorplasmid

Fluorescence Microscopy of Target Cells Demonstrates GOI Expression

Qualitative analysis of mVenus expression by fluorescence microscopy was conducted using Axio Observer Z1 showing that transduced HT1080 cells and non-transduced cells could be easily distinguished. In Figure 6 cells were excited with 505nm and fluorescence emission at 536nm was detected. Therefore, successful infection of tumor cells by recombinant viral particles carrying the assembled vectorplasmid coding for mVenus could be demonstrated.

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Figure 6: Fluorescence microscopy (Exciatation: 505nm, Emission: 536nm) was performed for detection of transduced cell expression mVenus. A:Cells detected in bright field picture B: Detection of mVenus expression can be observed.

 

Analysis of Target Cells by Flow Cytometry demonstrates GOI Expression

Characterizing the function of the hGH terminator, the beta-globin intron and the complete plasmid, several approaches were conducted followed by analysis via flow cytometry.

Influence of hGH terminator BioBrick on GOI Expression

The iGEM team Freiburg provides the hGH plolyadenylation sequence within the ‘Virus Construction Kit’ due to the fact that almost every eukaryotic mRNA is processed at their 3´ and 5´end except for histone mRNAs (Millevoi et al. 2006). Pre-mRNAs contain two canonical conserved sequences. First, the polyadenylation signal “AATAAA” which is recognized by the multiprotein complex and second the GT-rich region (downstream sequence element, DSE) which is located 30 nucleotides downstream of the cleavage site. The assembled 3´end-processing machinery cleaves the mRNA transcript immediately after a CA-nucleotide therefore defining the cleavage site (Danckwardt et al. 2008). Recombinant vectorplasmids were engineered containing the inverted terminal repeats (ITRs), a strong eukaryotic promoter (CMV promoter: BBa_K404102) and mVenus as gene of interest with and without the hGH terminator signal. Transduction of HT1080 cells with constant volume of viral particles containing the vectorplasmids and measuring mVenus expression 24-hours post infection by flow cytometry demonstrated that transgene expression of the constructs lacking the hGH termination signal is significantly reduced as shown in Figure 7 and Figure 8 confirming the expected results that hGH is essential for mRNA processing. The iGEM team Freiburg_Bioware 2010 therefore suggests using the provided hGH termination signal within the Virus Construction Kit for optimal gene expression.

Vectorplasmid lacking hGH termination signal

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Vectorplasmid containing hGH terminator signal

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Figure 7: Flow cytometry analysis of vectorplasmids with and without hGH terminator.

A: Gating non transduced cells (control); subcellular debris and clumps can be distinguished from single cells by size, estimated forward scatter (FS Lin) and granularity, estimated side scatter (SS Lin) B: Non transduced cells applied against mVenus (Analytical gate was set such that 1% or fewer of negative control cells fell within the positive region (R5). C: Gating transduced cells (R2 R14) (used plasmids for transfection: GOI: pSB1C3_lITR_CMV_beta-globin intron_mVenus_rITR (BBa_K404127), pHelper, pRC). D: Transduced cells plotted against mVenus, R10 comprises transduced cells by detecting mVenus expression. E: Overlay of non-transduced (red) and transduced (green) cells applied against mVenus.F: Gating non-transduced cells (control) G: Non-transduced cells applied against mVenus. H: Gating transduced cells (R2 R14) (used plasmids for transfection: GOI: reassembled pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (BBa_K404119), pHelper, pRC). I: Transduced cells applied against mVenus, R10 comprised transduced cells, by detecting mVenus expression. J: Overlay of non-transduced (red) and transduced (green) cells applied against mVenus.

 

 

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Figure 8: Flow cytometry analysis of vectorplasmids with and without hGH terminator. YFP expression of viral genomes was determined by flow cytomery after 24-hour post infection. Results demonstrate that mVenus expression of vectorplasmids lacking the hGH terminator is reduced significantly proving that the polyadenylation signal is essential for viral gene expression using recombinant viral vectors engineered by using components of the Virus Construction Kit.

 

Influence of Beta-globin intron Biobrick on GOI Expression

Providing an element assumed to be an enhancer of transgene expression (Nott et al. 2003), the iGEM team Freiburg tested a beta-globin intron derived from the human beta globin gene which can be fused upstream of the desired gene of interest. The beta-globin intron BioBrick consists of a partial chimeric CMV promoter followed by the intron II of the beta-globin gene. The 3´end of the intron is fused to the first 25 bases of human beta globin gene exon 3. The beta globin intron BioBrick is assumed to enhance eukaryotic gene expression (Nott et al. 2003). Analysis was conducted as described for the hGH terminator experiment (see above). As shown in Figure 9 and Figure 10 the vectorplasmid missing the beta-globin intron showed a negligible difference in mVenus expression compared to viral genomes containing the beta-globin intron. Considering these results and taking into account that a constant volume of viral particles has been used for transduction, the difference between the construct containing and lacking the beta-globin intron is minimal. Since packaging efficiency of the AAV-2 decreases with increasing sizes of the insert (Dong et al. 1996), the iGEM team Freiburg_Bioware suggests using the beta-globin intron in dependence on the size of your transgene.

Vectorplasmid lacking beta-globin intron

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Vectorplasmid containing beta-globin intron

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Figure 9: Flow cytometry analysis of vectorplasmids with and without beta-globin intron.  A: Gating non transduced cells (control); subcellular debris and clumps can be distinguished from single cells by size, estimated forward scatter (FS Lin) and granularity, estimated side scatter (SS Lin) B: Non transduced cells applied against mVenus (Analytical gate was set such that 1% or fewer of negative control cells fell within the positive region (R5). C: Gating transduced cells (R2 R14) (used plasmids for transfection: GOI: pSB1C3_lITR_CMV_mVenus_hGH_rITR (BBa_K404128), pHelper, pRC). D: Transduced cells plotted against mVenus, R10 comprised transduced cells, by detecting mVenus expression E: Overlay of non-transduced (red) and transduced (green) cells applied against mVenus F: Gating non-transduced cells (control). G: Non-transduced cells applied against mVenus (R5).H: Gating transduced cells (R2 R14) (used plasmids for transfection: GOI: reassembled pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (BBa_K404119), pHelper, pRC). I: Transduced cells applied against mVenus, R10 comprised transduced cells, by detecting mVenus expression. J: Overlay of non-transduced (red) and transduced (green) cells applied against mVenus.

 

 

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Figure 10: Flow cytometry analysis of vectorplasmids with and without beta-globin intron. 48-hours post transfection, viral particles were harvested by freeze-thaw lysis and centrifugation followed by HT1080 transduction. YFP expression of vectorplasmids was determined by flow cytometry 24-hours post infection. The vectorplasmid missing the beta-globin intron showed a negligible difference in mVenus expression compared to viral plasmid containing the beta-globin intron.

 

Functionality of the Full Assembled Vectorplasmid Demonstrated by GOI Expression

After assembly of plasmids containing all required elements (see Figure 1), functionality was tested in cell culture. AAV-293 cells stably expressing E1A and E1B proteins were transfected with three plasmids  (pHelper, pRC, pGOI). Virus particles were harvested 72-hours post-transfection and the tumor cell line HT1080 was transduced with the recombinant viral vectors encapsidating the gene of interest mVenus (BBa_I757008).

The iGEM team Freiburg_Bioware 2010 compared the standard-plasmid containing a subcloned mVenus (pAAV_mVenus, derived from the Stratagene system) with the assembled plasmid pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (pSB1C3_mVenus: BBa_K404119). Fluorescence expression data obtained by flow cytometry analysis are shown in Figure 11 and Figure 12. Comparing mVenus expression of the standard plasmid and the modified, assembled plasmid reveals that biological functionality of the reassembled plasmid was confirmed.

pSB1C3_mVenus (BBa_K404119)

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pAAV_mVenus (Stratagene)

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Figure 11: Flow cytometry analysis of reassembled vectorplasmid (BBa_K404119) compared to standard plasmid provided by Stratagene. A: Gating non transduced cells (control); subcellular debris and clumps can be distinguished from single cells by size, estimated forward scatter (FS Lin) and granularity, estimated side scatter (SS Lin) B: Non transduced cells plotted against mVenus (Analytical gate was set such that 1% or fewer of negative control cells fell within the positive region (R5).C: Gating transduced cells (R2 R14) (used plasmids for transfection: pGOI: pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (pSB1C3_mVenus: BBa_K404119), pHelper, pRC. D: Transduced cells plotted against mVenus, R10 comprised transduced cells, by detecting mVenus expression. E: Overlay of non-transduced (red) and transduced (green). F: Gating non transduced cells (control). G: Non-transduced cells plotted against mVenus (R5). H: Gating transduced cells (R14 R2) (used plasmids for transfection: pGOI: pAAV_mVenus, pHelper). I: Transduced cells plotted against mVenus, R10 comprised transduced cells, by detecting mVenus expression. J: Overlay of non-transduced (red) and transduced (green) cells plotted against mVenus expression.

 

 

 

 

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Figure 12: Flow cytometry analysis of reassembled vectorplasmid (BBa_K404119) compared to standard plasmid provided by Stratagene. Fluorescence of the standard plasmid pAAV_mVenus (Stratagene) and the recombinant pSB1C3_mVenus (BBa_K404119) construct was measured. As demonstrated mVenus expression is enhanced in the assembled plasmid (pSB1C3_mVenus) compared to the standard pAAV_mVenus construct.

 

Danckwardt, S., Hentze, M.W. & Kulozik, A.E., 2008. 3’ end mRNA processing: molecular mechanisms and implications for health and disease. The EMBO journal, 27(3), pp.482-98. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18256699.

Dong, J.Y., Fan, P.D. & Frizzell, R.A., 1996. Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Human gene therapy, 7(17), pp.2101-12. Available at: http://www.ncbi.nlm.nih.gov/pubmed/8934224.

Millevoi, S. et al., 2006. An interaction between U2AF 65 and CF I(m) links the splicing and 3’ end processing machineries. The EMBO journal, 25(20), pp.4854-64. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17024186.

Nott, A., Meislin, S.H. & Moore, M.J., 2003. A quantitative analysis of intron effects on mammalian gene expression. RNA (New York, N.Y.), 9(5), pp.607-17. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12702819.