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

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<html>
<html>
-
<div
+
<h1>Modularization</h1>
-
style="border-style: none none solid; border-color: -moz-use-text-color -moz-use-text-color windowtext; border-width: medium medium 1pt; padding: 0cm 0cm 1pt;">
+
<br>
-
<p class="MsoTocHeading">Contents</p>
+
<h2>Contents</h2>
-
</div>
+
<h3>Modularization of the vectorplasmid</h3>
-
<p class="MsoToc2"><span class="MsoHyperlink"><a href="#_Toc275800680"><span
+
<p class="MsoToc2"><a href="#_Toc275800680"><span lang="EN-US">Introduction
-
lang="EN-US">Introduction to Modularization of Vectorplasmid</span><span
+
to
-
style="color: windowtext; display: none; text-decoration: none;">. </span><span
+
Modularization of Vectorplasmid</span><span
-
style="color: windowtext; display: none; text-decoration: none;">1</span></a></span></p>
+
style="color: windowtext; display: none; text-decoration: none;">.
-
<p class="MsoToc3"><span class="MsoHyperlink"><a href="#_Toc275800681"><span
+
</span><span
-
lang="EN-US">Recombinant and Modular Vectorplasmid Carrying GOI</span><span
+
style="color: windowtext; display: none; text-decoration: none;">1</span></a></p>
-
style="color: windowtext; display: none; text-decoration: none;"> </span><span
+
<p class="MsoToc3"><a href="#_Toc275800681"><span lang="EN-US">Recombinant
-
style="color: windowtext; display: none; text-decoration: none;">2</span></a></span></p>
+
and
-
<p class="MsoToc3"><span class="MsoHyperlink"><a href="#_Toc275800682"><span
+
Modular Vectorplasmid Carrying GOI</span><span
-
lang="EN-US">Cloning and Combination Strategies for the Vectorplasmid</span><span
+
style="color: windowtext; display: none; text-decoration: none;">
-
style="color: windowtext; display: none; text-decoration: none;">. </span><span
+
</span><span
-
style="color: windowtext; display: none; text-decoration: none;">3</span></a></span></p>
+
style="color: windowtext; display: none; text-decoration: none;">2</span></a></p>
-
<p class="MsoToc3"><span class="MsoHyperlink"><a href="#_Toc275800683"><span
+
<p class="MsoToc3"><a href="#_Toc275800682"><span lang="EN-US">Cloning
-
lang="EN-US">Testing functionality of Assembled Vectorplasmid</span><span
+
and
-
style="color: windowtext; display: none; text-decoration: none;">. </span><span
+
Combination Strategies for the Vectorplasmid</span><span
-
style="color: windowtext; display: none; text-decoration: none;">7</span></a></span></p>
+
style="color: windowtext; display: none; text-decoration: none;">.
-
<p class="MsoToc4"><span class="MsoHyperlink"><a href="#_Toc275800684"><span
+
</span><span
-
lang="EN-US">Fluorescence Microscopy of Target Cells Demonstrates GOI
+
style="color: windowtext; display: none; text-decoration: none;">3</span></a></p>
-
Expression</span><span
+
<p class="MsoToc3"><a href="#_Toc275800683"><span lang="EN-US">Testing
-
style="color: windowtext; display: none; text-decoration: none;">. </span><span
+
functionality of Assembled Vectorplasmid</span><span
-
style="color: windowtext; display: none; text-decoration: none;">7</span></a></span></p>
+
style="color: windowtext; display: none; text-decoration: none;">.
-
<p class="MsoToc4"><span class="MsoHyperlink"><a href="#_Toc275800685"><span
+
</span><span
-
lang="EN-US">Analysis of Target Cells by Flow Cytometry demonstrates
+
style="color: windowtext; display: none; text-decoration: none;">7</span></a></p>
-
GOI Expression</span><span
+
<p class="MsoToc4"><a href="#_Toc275800684"><span lang="EN-US">Fluorescence
-
style="color: windowtext; display: none; text-decoration: none;">. </span><span
+
Microscopy of Target Cells Demonstrates GOI Expression</span><span
-
style="color: windowtext; display: none; text-decoration: none;">8</span></a></span></p>
+
style="color: windowtext; display: none; text-decoration: none;">.
-
<p class="MsoToc5"><span class="MsoHyperlink"><a href="#_Toc275800686"><span
+
</span><span
-
lang="EN-US">Influence of hGH terminator BioBrick on GOI Expression</span><span
+
style="color: windowtext; display: none; text-decoration: none;">7</span></a></p>
-
style="color: windowtext; display: none; text-decoration: none;">. </span><span
+
<p class="MsoToc4"><a href="#_Toc275800685"><span lang="EN-US">Analysis
-
style="color: windowtext; display: none; text-decoration: none;">8</span></a></span></p>
+
of Target
-
<p class="MsoToc5"><span class="MsoHyperlink"><a href="#_Toc275800687"><span
+
Cells by Flow Cytometry demonstrates GOI Expression</span><span
-
lang="EN-US">Influence of <i>Beta-globin</i> intron Biobrick on GOI
+
style="color: windowtext; display: none; text-decoration: none;">.
-
Expression</span><span
+
</span><span
-
style="color: windowtext; display: none; text-decoration: none;">. </span><span
+
style="color: windowtext; display: none; text-decoration: none;">8</span></a></p>
-
style="color: windowtext; display: none; text-decoration: none;">11</span></a></span></p>
+
<p class="MsoToc5"><a href="#_Toc275800686"><span lang="EN-US">Influence
-
<p class="MsoToc5"><span class="MsoHyperlink"><a href="#_Toc275800688"><span
+
of hGH
-
lang="EN-US">Functionality of the Full Assembled Vectorplasmid
+
terminator BioBrick on GOI Expression</span><span
-
Demonstrated by
+
style="color: windowtext; display: none; text-decoration: none;">.
-
GOI Expression</span><span
+
</span><span
-
style="color: windowtext; display: none; text-decoration: none;">. </span><span
+
style="color: windowtext; display: none; text-decoration: none;">8</span></a></p>
-
style="color: windowtext; display: none; text-decoration: none;">14</span></a></span></p>
+
<p class="MsoToc5"><a href="#_Toc275800687"><span lang="EN-US">Influence
-
<p class="MsoToc3"><span class="MsoHyperlink"><a href="#_Toc275800689"><span
+
of <i>Beta-globin</i>
-
lang="EN-US">Conclusion</span><span
+
intron Biobrick on GOI Expression</span><span
-
style="color: windowtext; display: none; text-decoration: none;">. </span><span
+
style="color: windowtext; display: none; text-decoration: none;">.
-
style="color: windowtext; display: none; text-decoration: none;">16</span></a></span></p>
+
</span><span
-
<p class="MsoToc3"><span class="MsoHyperlink"><a href="#_Toc275800690"><span
+
style="color: windowtext; display: none; text-decoration: none;">11</span></a></p>
-
lang="EN-US">References</span><span
+
<p class="MsoToc5"><a href="#_Toc275800688"><span lang="EN-US">Functionality
-
style="color: windowtext; display: none; text-decoration: none;">. </span><span
+
of the
-
style="color: windowtext; display: none; text-decoration: none;">17</span></a></span></p>
+
Full Assembled Vectorplasmid Demonstrated by GOI Expression</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">14</span></a></p>
 +
<p class="MsoToc3"><a href="#_Toc275800689"><span lang="EN-US">Conclusion</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">16</span></a></p>
 +
<p class="MsoToc3"><a href="#_Toc275800690"><span lang="EN-US">References</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">17</span></a></p>
<p class="MsoNormal"><a name="_Toc275797953"><b>&nbsp;</b></a></p>
<p class="MsoNormal"><a name="_Toc275797953"><b>&nbsp;</b></a></p>
-
<h2 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800680">Introduction
+
<h3>Modularization of the RepVP123 plasmids</h3>
-
to Modularization of Vectorplasmid</a></h2>
+
<div class="WordSection1">
-
<p class="MsoNormal"><span lang="EN-US">Producing recombinant virus
+
<p class="MsoToc2"><a href="#_Toc275885921"><span lang="EN-US">Overview
 +
of RepVP123
 +
plasmid</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">2</span></a></p>
 +
<p class="MsoToc2"><a href="#_Toc275885922"><span lang="EN-US">Modularization:
 +
Overview</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">..
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">2</span></a></p>
 +
<p class="MsoToc3"><a href="#_Toc275885923"><span lang="EN-US">Modularization:
 +
Removing iGEM restriction sites and establishing loop insertion
 +
capability</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">3</span></a></p>
 +
<p class="MsoToc4"><a href="#_Toc275885924"><span lang="EN-US">Modifications
 +
in Rep</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">3</span></a></p>
 +
<p class="MsoToc4"><a href="#_Toc275885925"><span lang="EN-US">Modifications
 +
in
 +
VP123</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">4</span></a></p>
 +
<p class="MsoToc3"><a href="#_Toc275885926"><span lang="EN-US">Modularization:
 +
Adapting pSB1C3 to loop insertions – pSB1C3_001</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">7</span></a></p>
 +
<p class="MsoToc4"><a href="#_Toc275885927"><span lang="EN-US">Turning-off
 +
natural
 +
tropism: HSPG-knock-out</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">.
 +
</span><span
 +
style="color: windowtext; display: none; text-decoration: none;">9</span></a></p>
 +
<p class="MsoToc2" style="margin-left: 0cm; text-indent: 0cm;">&nbsp;</p>
 +
<span
 +
style="font-size: 11pt; line-height: 115%; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
 +
lang="EN-US"><br style="page-break-before: always;" clear="all">
 +
</span>
 +
<h2><a name="_Toc275800680">Introduction to
 +
Modularization of vector
 +
plasmid</a></h2>
 +
<br>
 +
<p class="MsoNormal"><span lang="EN-US">Producing
 +
recombinant virus
particles for
particles for
therapeutical means is, besides specifically target cells, purification
therapeutical means is, besides specifically target cells, purification
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to match
to match
the iGEM standard. Functional activity was tested in cell culture.</span></p>
the iGEM standard. Functional activity was tested in cell culture.</span></p>
-
<p class="MsoNormal"><span lang="EN-US">Differing from the wildtype
+
<p class="MsoNormal"><span lang="EN-US">Differing
 +
from the wildtype
AAV-2 genome,
AAV-2 genome,
the Helper Free System provided by Stratagene comprises three plasmids
the Helper Free System provided by Stratagene comprises three plasmids
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another helper plasmid (pHelper), which provides the necessary proteins
another helper plasmid (pHelper), which provides the necessary proteins
normally obtained by superinfection with helper viruses such as
normally obtained by superinfection with helper viruses such as
-
adenovirus or
+
adenovirus or herpes
-
herpes simplex virus. These helper genes are required for full viral
+
simplex virus. These helper genes are required for full viral assembly
-
assembly
+
by
-
by regulating gene expression of Rep and Cap proteins.</span></p>
+
regulating gene expression of Rep and Cap proteins.</span></p>
<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800681"></a><a
<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800681"></a><a
-
name="_Toc275797954">Recombinant and Modular Vectorplasmid Carrying
+
name="_Toc275797954"><span lang="EN-US"><span
-
GOI</a></h3>
+
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"></span></span><span
-
<p class="MsoNormal"><span lang="EN-US">The iGEM team Freiburg_Bioware
+
lang="EN-US">Recombinant and Modular Vectorplasmid Carrying
 +
GOI</span></a></h3>
 +
<p class="MsoNormal"><span lang="EN-US">The
 +
iGEM team Freiburg_Bioware
2010 provides
2010 provides
a modular Virus Construction Kit for therapeutical applications,
a modular Virus Construction Kit for therapeutical applications,
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enhancer element and the hGH terminator was PCR amplified and fused
enhancer element and the hGH terminator was PCR amplified and fused
together <i>de
together <i>de
-
novo</i>.</span><span lang="EN-US"> As shown in </span><span
+
novo</i>.</span><span lang="EN-US"> As shown
-
lang="EN-US">Figure 1</span><span lang="EN-US">, the vectorplasmid was
+
in </span><span lang="EN-US">Figure 1</span><span lang="EN-US">, the
 +
vectorplasmid was
assembled
assembled
with the produced BioBricks consisting of the left and right ITR
with the produced BioBricks consisting of the left and right ITR
-
(BBa_K404100
+
(<a href = "http://partsregistry.org/Part:BBa_K404100" target="blank" > BBa_K404100</a>
-
and BBa_K404101), a promoter (pCMV :BBa_K404102 or phTERT:
+
and <a href = "http://partsregistry.org/Part:BBa_K404101" target="blank" > BBa_K40410</a>), a promoter (pCMV :<a href = "http://partsregistry.org/Part:BBa_K404102" target="blank" > BBa_K404102</a> or phTERT:
-
BBa_K404106)) , the
+
<a href = "http://partsregistry.org/Part:BBa_K404106" target="blank" > BBa_K404106</a>)) , the
-
beta-globin intron (BBa_K404107), the gene of interests (fluorescent
+
beta-globin intron (<a href = "http://partsregistry.org/Part:BBa_K404107" target="blank" > BBa_K404107</a>), the gene of interests (fluorescent
proteins
proteins
-
mVenus: BBa_I757008 and mCherry: BBa_J06504, suicide genes mGMK_TK30:
+
mVenus: <a href = "http://partsregistry.org/Part:BBa_I757008" target="blank" > BBa_I757008</a> and mCherry: <a href = "http://partsregistry.org/Part:BBa_J06504" target="blank" > BBa_J06504</a>, suicide genes mGMK_TK30:
-
BBa_K404112,
+
(<a href = "http://partsregistry.org/Part:BBa_K404112" target="blank" > BBa_K404112</a>,
-
mGMK_SR39: BBa_K404315 and CD: BBa_K404112) and the hGH terminator
+
mGMK_SR39: <a href = "http://partsregistry.org/Part:BBa_K4043150" target="blank" > BBa_K404315</a> and CD: <a href = "http://partsregistry.org/Part:BBa_K404112" target="blank" > BBa_K404112</a>) and the hGH terminator
-
(BBa_K404108).</span></p>
+
(<a href = "http://partsregistry.org/Part:BBa_K404108" target="blank" > BBa_K404108</a>).</span></p>
<div align="center">
<div align="center">
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; border-collapse: collapse;" border="1"
+
style="border: medium none ; border-collapse: collapse;" border="0"
cellpadding="0" cellspacing="0">
cellpadding="0" cellspacing="0">
<tbody>
<tbody>
<tr style="height: 522.25pt;">
<tr style="height: 522.25pt;">
-
<td
+
<td style="padding: 0cm 5.4pt; width: 441.75pt; height: 522.25pt;"
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 441.75pt; height: 522.25pt;"
+
valign="top" width="589">
valign="top" width="589">
<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 5" src="Freiburg10_Modularization_GOI-Dateien/image001.jpg"
+
id="Grafik 5"
 +
src="https://static.igem.org/mediawiki/2010/a/ae/Freiburg10_Overview_BBa_Vectorplasmid.png"
height="692" width="580"></p>
height="692" width="580"></p>
<p class="MsoCaption" style="text-indent: 0cm;"><span lang="EN-US">Figure
<p class="MsoCaption" style="text-indent: 0cm;"><span lang="EN-US">Figure
</span><span lang="EN-US">1</span><span lang="EN-US">: </span><span
</span><span lang="EN-US">1</span><span lang="EN-US">: </span><span
-
style="font-weight: normal;" lang="EN-US">Overview of the theoretical
+
style="font-weight: normal;" lang="EN-US">Overview of
 +
the theoretical
sequence of each BioBrick provided within the Virus Construction Kit
sequence of each BioBrick provided within the Virus Construction Kit
for an intact and fully functional rAAV genome. The plasmid in the
for an intact and fully functional rAAV genome. The plasmid in the
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and Combination Strategies for the Vectorplasmid</span></a><span
and Combination Strategies for the Vectorplasmid</span></a><span
lang="EN-US"> </span></h3>
lang="EN-US"> </span></h3>
-
<p class="MsoNormal"><span lang="EN-US">Organization of the recombinant
+
<p class="MsoNormal"><span lang="EN-US">Organization
 +
of the recombinant
viral DNA was
viral DNA was
modified ensuring several layers of specificity to our systems
modified ensuring several layers of specificity to our systems
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In
In
order to modularize the rAAV sequence, each plasmid element (</span><span
order to modularize the rAAV sequence, each plasmid element (</span><span
-
lang="EN-US">Figure 1</span><span lang="EN-US">) was PCR-amplified and
+
lang="EN-US">Figure 1</span><span lang="EN-US">)
 +
was PCR-amplified and
cloned into
cloned into
the iGEM standard plasmid pSB1C3. Furthermore, the iGEM team
the iGEM standard plasmid pSB1C3. Furthermore, the iGEM team
Freiburg_Bioware
Freiburg_Bioware
2010 performed three site-directed mutagenesis in the gene of interest
2010 performed three site-directed mutagenesis in the gene of interest
-
TK30 (BBa_K404109)
+
TK30 (<a href = "http://partsregistry.org/Part:BBa_K404109" target="blank" > BBa_K404109</a>)
and cytosine deaminase (</span><span
and cytosine deaminase (</span><span
-
style="font-size: 9pt; line-height: 200%;" lang="EN-US">BBa_K404112</span><span
+
style="font-size: 9pt; line-height: 200%;" lang="EN-US"><a href = "http://partsregistry.org/Part:BBa_K404112" target="blank" > BBa_K404112</a></span><span
lang="EN-US">) for deletion of PstI and
lang="EN-US">) for deletion of PstI and
NgoMIV iGEM site (for further information see the results page of
NgoMIV iGEM site (for further information see the results page of
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Strategy for ITRs’).</span></p>
Strategy for ITRs’).</span></p>
<p class="MsoNormal"><span lang="EN-US">In </span><span lang="EN-US">Figure
<p class="MsoNormal"><span lang="EN-US">In </span><span lang="EN-US">Figure
-
2</span><span lang="EN-US"> the schematic overview of the
+
2</span><span lang="EN-US"> the schematic overview
 +
of the
modularization process can be seen which has been followed to conduct
modularization process can be seen which has been followed to conduct
the
the
Line 199: Line 271:
<div align="center">
<div align="center">
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; border-collapse: collapse;" border="1"
+
style="border: medium none ; border-collapse: collapse;" border="0"
cellpadding="0" cellspacing="0">
cellpadding="0" cellspacing="0">
<tbody>
<tbody>
<tr style="height: 26.15pt;">
<tr style="height: 26.15pt;">
-
<td
+
<td style="padding: 0cm 5.4pt; width: 340.15pt; height: 26.15pt;"
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 340.15pt; height: 26.15pt;"
+
valign="top" width="454">
valign="top" width="454">
<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-Dateien/image002.gif"
+
id="Grafik 2"
 +
src="https://static.igem.org/mediawiki/2010/3/39/Freiburg10_Overview_Assembly_GOI.png"
alt="Beschreibung: http://partsregistry.org/wiki/images/1/1c/Freiburg10_Vectorplasmid_cloning.png"
alt="Beschreibung: http://partsregistry.org/wiki/images/1/1c/Freiburg10_Vectorplasmid_cloning.png"
height="272" width="439"></p>
height="272" width="439"></p>
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</div>
</div>
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
-
<p class="MsoNormal"><span lang="EN-US">The iGEM team Freiburg_Bioware
+
<p class="MsoNormal"><span lang="EN-US">The
 +
iGEM team Freiburg_Bioware
provides two
provides two
examples demonstrating the assembly procedure for constructing
examples demonstrating the assembly procedure for constructing
Line 237: Line 310:
shown in </span><span lang="EN-US">Figure 3</span><span lang="EN-US">
shown in </span><span lang="EN-US">Figure 3</span><span lang="EN-US">
the theoretical cloning performed
the theoretical cloning performed
-
for assembling the BioBricks <i>beta-globin </i>intron and
+
for assembling the BioBricks <i>beta-globin </i>intron
 +
and
leftITR_CMV together
leftITR_CMV together
can be observed. </span></p>
can be observed. </span></p>
<div align="center">
<div align="center">
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; border-collapse: collapse;" border="1"
+
style="border: medium none ; border-collapse: collapse;" border="0"
cellpadding="0" cellspacing="0">
cellpadding="0" cellspacing="0">
<tbody>
<tbody>
<tr style="height: 23.95pt;">
<tr style="height: 23.95pt;">
-
<td
+
<td style="padding: 0cm 5.4pt; width: 349.9pt; height: 23.95pt;"
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 349.9pt; height: 23.95pt;"
+
valign="top" width="467">
valign="top" width="467">
<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-Dateien/image003.gif"
+
id="Grafik 74"
 +
src="https://static.igem.org/mediawiki/2010/e/e3/Freiburg10_TheoreticalCloning_ITR_CMV_beta.png"
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
name="_Ref275783160"><span lang="EN-US">Figure </span></a><span
name="_Ref275783160"><span lang="EN-US">Figure </span></a><span
lang="EN-US">4</span><span lang="EN-US">:</span><span lang="EN-US"> </span><span
lang="EN-US">4</span><span lang="EN-US">:</span><span lang="EN-US"> </span><span
-
style="font-weight: normal;" lang="EN-US">Theoretical cloning of the
+
style="font-weight: normal;" lang="EN-US">Theoretical
-
composite part leftITR_CMV to the <i>beta-globin</i> intron BioBrick
+
cloning of the
-
leading to the plasmid leftITR_CMV_<i>beta-globin</i> intron.</span></p>
+
composite part leftITR_CMV to the <i>beta-globin</i>
 +
intron BioBrick
 +
leading to the plasmid leftITR_CMV_<i>beta-globin</i>
 +
intron.</span></p>
</td>
</td>
</tr>
</tr>
Line 265: Line 342:
</div>
</div>
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
-
<p class="MsoNormal"><span lang="EN-US">The plasmids were digested with
+
<p class="MsoNormal"><span lang="EN-US">The
 +
plasmids were digested with
both XbaI
both XbaI
and PstI (beta-globin intron: </span><span
and PstI (beta-globin intron: </span><span
-
style="font-size: 9pt; line-height: 200%; color: black;" lang="EN-US">BBa_K404107</span><span
+
style="font-size: 9pt; line-height: 200%; color: black;" lang="EN-US"><a href = "http://partsregistry.org/Part:BBa_K404107" target="blank" > BBa_K404107</a></span><span
-
lang="EN-US">) or SpeI and
+
lang="EN-US">)
 +
or SpeI and
PstI (leftITR_CMV) and loaded on an agarose gel. As demonstrated in the
PstI (leftITR_CMV) and loaded on an agarose gel. As demonstrated in the
-
preparative
+
preparative gel in </span><span lang="EN-US">Figure
-
gel in </span><span lang="EN-US">Figure 4</span><span lang="EN-US">,
+
4</span><span lang="EN-US">,
-
the expected
+
the expected bands could be detected under UV light and the extracted
-
bands could be detected under UV light and the extracted DNA could be
+
DNA could
-
successfully ligated. Each assembly step for producing BioBrick
+
be successfully ligated. Each assembly step for producing BioBrick
-
intermediates
+
intermediates was conducted following the same strategy.</span></p>
-
was conducted following the same strategy.</span></p>
+
<div align="center">
<div align="center">
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; border-collapse: collapse;" border="1"
+
style="border: medium none ; border-collapse: collapse;" border="0"
cellpadding="0" cellspacing="0">
cellpadding="0" cellspacing="0">
<tbody>
<tbody>
<tr style="height: 129.95pt;">
<tr style="height: 129.95pt;">
-
<td
+
<td style="padding: 0cm 5.4pt; width: 312.75pt; height: 129.95pt;"
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 312.75pt; height: 129.95pt;"
+
valign="top" width="417">
valign="top" width="417">
<p class="MsoNormal"
<p class="MsoNormal"
Line 293: Line 370:
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 id="Grafik 77"
-
src="Freiburg10_Modularization_GOI-Dateien/image004.gif"
+
src="https://static.igem.org/mediawiki/2010/4/49/Freiburg10_Cloning_Intermediate_GOI.png"
alt="Beschreibung: \\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="Beschreibung: \\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>
Line 301: Line 378:
promoters. </span><span style="font-weight: normal;" lang="EN-US">Fusion
promoters. </span><span style="font-weight: normal;" lang="EN-US">Fusion
of the BioBrick part <i>beta-globin</i> (</span><span
of the BioBrick part <i>beta-globin</i> (</span><span
-
style="color: black;" lang="EN-US">BBa_K404107</span><span
+
style="color: black;" lang="EN-US"><a href = "http://partsregistry.org/Part:BBa_K404107" target="blank" > BBa_K404107</a></span><span
-
style="font-weight: normal;" lang="EN-US">) intron to the composite
+
style="font-weight: normal;" lang="EN-US">) intron to
 +
the composite
parts leftITR_pCMV and leftITR_phTERT, respectively, was performed
parts leftITR_pCMV and leftITR_phTERT, respectively, was performed
following the BioBrick assembly strategy by digesting the insert with
following the BioBrick assembly strategy by digesting the insert with
Line 318: Line 396:
</div>
</div>
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
-
<p class="MsoNormal"><span lang="EN-US">Separated fragments were
+
<p class="MsoNormal"><span lang="EN-US">Separated
 +
fragments were
extracted using
extracted using
the Gel Extraction Kit provided by Qiagen (Hilden, Germany) and ligated
the Gel Extraction Kit provided by Qiagen (Hilden, Germany) and ligated
with
with
-
T4-ligase. After ligation has been carried out, <i>E. coli</i> XL-1B
+
T4-ligase. After ligation has been carried out, <i>E. coli</i>
 +
XL-1B
cells were
cells were
transformed and incubated over night at 37°C. Picking clones from the
transformed and incubated over night at 37°C. Picking clones from the
Line 330: Line 410:
revealed that cloning was successful obtaining the composite part
revealed that cloning was successful obtaining the composite part
leftITR_CMV_<i>beta-globin</i>
leftITR_CMV_<i>beta-globin</i>
-
intron (BBa_K404117).</span></p>
+
intron (<a href = "http://partsregistry.org/Part:BBa_K404117" target="blank" > BBa_K404117</a>).</span></p>
-
<p class="MsoNormal"><span lang="EN-US">Plasmid production
+
<p class="MsoNormal"><span lang="EN-US">Plasmid
 +
production
incorporating all
incorporating all
required elements for transgene expression and genome encapsidation
required elements for transgene expression and genome encapsidation
Line 340: Line 421:
gene of
gene of
interest and the promoter fused to the left ITR. </span><span
interest and the promoter fused to the left ITR. </span><span
-
lang="EN-US">Figure 5</span><span lang="EN-US"> demonstrates the
+
lang="EN-US">Figure 5</span><span lang="EN-US">
 +
demonstrates the
assembly performed
assembly performed
-
with pSB1C3_leftITR_phTERT_<i>beta-globin</i> intron_mVenus and
+
with pSB1C3_leftITR_phTERT_<i>beta-globin</i> intron_mVenus
-
pSB1C3_hGH_rightITR (BBa_K404116). The fragment obtained after
+
and
 +
pSB1C3_hGH_rightITR (<a href = "http://partsregistry.org/Part:BBa_K404116" target="blank" > BBa_K404116</a>). The fragment obtained after
digestion on the
digestion on the
left lane fits to the hGH-terminator_rightITR length. The isolated
left lane fits to the hGH-terminator_rightITR length. The isolated
Line 351: Line 434:
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><span style="line-height: 200%; color: black;" lang="EN-US">BBa_K404124</span><span
+
(</span><span style="line-height: 200%; color: black;" lang="EN-US"><a href = "http://partsregistry.org/Part:BBa_K404124" target="blank" > BBa_K404124</a></span><span
-
lang="EN-US">). </span></p>
+
lang="EN-US">).
 +
</span></p>
<div align="center">
<div align="center">
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; border-collapse: collapse;" border="1"
+
style="border: medium none ; border-collapse: collapse;" border="0"
cellpadding="0" cellspacing="0">
cellpadding="0" cellspacing="0">
<tbody>
<tbody>
<tr style="height: 136.9pt;">
<tr style="height: 136.9pt;">
-
<td
+
<td style="padding: 0cm 5.4pt; width: 308.4pt; height: 136.9pt;"
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 308.4pt; height: 136.9pt;"
+
valign="top" width="411">
valign="top" width="411">
<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 id="Grafik 80"
-
src="Freiburg10_Modularization_GOI-Dateien/image005.gif"
+
src="https://static.igem.org/mediawiki/2010/9/93/Freiburg10_Cloning_Full_GOI.png"
alt="Beschreibung: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\18.09_Cloning_Full_phTERT_mVenus.png"
alt="Beschreibung: \\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>
Line 371: Line 454:
name="_Ref275784510"><span lang="EN-US">Figure </span></a><span
name="_Ref275784510"><span lang="EN-US">Figure </span></a><span
lang="EN-US">6</span><span style="font-weight: normal;" lang="EN-US">:
lang="EN-US">6</span><span style="font-weight: normal;" lang="EN-US">:
-
</span><span lang="EN-US">Modularization of the assembled
+
</span><span lang="EN-US">Modularization of
 +
the assembled
vectorplasmid containing the phTERT promoter and mVenus as gene of
vectorplasmid containing the phTERT promoter and mVenus as gene of
interest.</span><span style="font-weight: normal;" lang="EN-US"> Fusion
interest.</span><span style="font-weight: normal;" lang="EN-US"> Fusion
Line 389: Line 473:
</div>
</div>
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
-
<p class="MsoNormal"><span lang="EN-US">Since cloning does not confirm
+
<p class="MsoNormal"><span lang="EN-US">Since
 +
cloning does not confirm
biological
biological
activity, we analyzed the plasmids and their functional components, hGH
activity, we analyzed the plasmids and their functional components, hGH
-
terminator and <i>beta-globin</i> intron, in cell culture. Assembled
+
terminator and <i>beta-globin</i> intron, in cell culture.
-
plasmids have
+
Assembled
-
been cotransfected, using AAV-293 cells, which provide the stable
+
plasmids
-
integrated
+
have been cotransfected, using AAV-293 cells, which provide the stable
-
E1A and E1B genes, with helper plasmids required for capsid assembly
+
integrated E1A and E1B genes, with helper plasmids required for capsid
-
and
+
assembly and genome encapsidation (pRC and pHelper) in a molar ratio of
-
genome encapsidation (pRC and pHelper) in a molar ratio of 1:1:1
+
1:1:1
(pGOI:pRC:pHelper). Virus particles containing the single stranded DNA
(pGOI:pRC:pHelper). Virus particles containing the single stranded DNA
were
were
-
harvested 72-hours post transfection and HT1080 cells transduced with
+
harvested 72 hours post transfection and HT1080 cells transduced with
constant
constant
-
volumes of viral vectors. 48-hours post infection; transduced cells
+
volumes of viral vectors. 48 hours post infection; transduced cells
expressing
expressing
the gene of interest were analyzed by flow cytometry. Facilitating and
the gene of interest were analyzed by flow cytometry. Facilitating and
Line 418: Line 503:
lang="EN-US">Fluorescence
lang="EN-US">Fluorescence
Microscopy of Target Cells Demonstrates GOI Expression</span></a></h4>
Microscopy of Target Cells Demonstrates GOI Expression</span></a></h4>
-
<p class="MsoNormal"><span lang="EN-US">Qualitative analysis of mVenus
+
<p class="MsoNormal"><span lang="EN-US">Qualitative
 +
analysis of mVenus
expression
expression
by fluorescence microscopy was conducted using Axio Observer Z1 showing
by fluorescence microscopy was conducted using Axio Observer Z1 showing
Line 431: Line 517:
assembled vectorplasmid
assembled vectorplasmid
coding for mVenus could be demonstrated. </span></p>
coding for mVenus could be demonstrated. </span></p>
 +
<div align="center">
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; border-collapse: collapse;" border="1"
+
style="border: medium none ; border-collapse: collapse;" border="0"
cellpadding="0" cellspacing="0">
cellpadding="0" cellspacing="0">
<tbody>
<tbody>
<tr>
<tr>
-
<td
+
<td style="padding: 0cm 5.4pt; width: 250.7pt;" valign="top"
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 250.7pt;"
+
width="334">
-
valign="top" width="334">
+
<p class="MsoNormal"
<p class="MsoNormal"
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
Line 444: Line 530:
<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 style="width: 264px; height: 218px;" id="Grafik 18"
-
src="Freiburg10_Modularization_GOI-Dateien/image006.jpg"
+
src="https://static.igem.org/mediawiki/2010/a/af/Freiburg10_Microscopy_Overlay_mVenus_1_GOI.png"
-
alt="Beschreibung: Freiburg10_2Transd30µg_unverd_2_(c1).JPG (1388×1040)"
+
alt=""></p>
-
height="218" width="264"></p>
+
</td>
</td>
-
<td
+
<td style="padding: 0cm 5.4pt; width: 230.4pt;" valign="top"
-
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0cm 5.4pt; width: 230.4pt;"
+
width="307">
-
valign="top" width="307">
+
<p class="MsoNormal"
<p class="MsoNormal"
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
Line 457: Line 541:
<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 style="width: 242px; height: 220px;" id="Grafik 19"
-
src="Freiburg10_Modularization_GOI-Dateien/image007.jpg"
+
src="https://static.igem.org/mediawiki/2010/f/fc/Freiburg10_Microscopy_Overlay_grey_1_GOI.png"
-
alt="Beschreibung: https://static.igem.org/mediawiki/2010/f/f1/Freiburg10_2Transd30%C2%B5g_unverd_%28c1%29.JPG"
+
alt=""></p>
-
height="220" width="242"></p>
+
</td>
</td>
</tr>
</tr>
<tr>
<tr>
-
<td
+
<td style="padding: 0cm 5.4pt; width: 250.7pt;" valign="top"
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 250.7pt;"
+
width="334">
-
valign="top" width="334">
+
<p class="MsoNormal"
<p class="MsoNormal"
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
Line 472: Line 554:
<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 style="width: 258px; height: 195px;" id="Grafik 16"
-
src="Freiburg10_Modularization_GOI-Dateien/image008.jpg"
+
src="https://static.igem.org/mediawiki/2010/4/48/Freiburg10_Microscopy_Overlay_mVenus_2_GOI.png"
-
alt="Beschreibung: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"
+
alt=""></p>
-
height="195" width="258"></p>
+
</td>
</td>
-
<td
+
<td style="padding: 0cm 5.4pt; width: 230.4pt;" valign="top"
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 230.4pt;"
+
width="307">
-
valign="top" width="307">
+
<p class="MsoNormal"
<p class="MsoNormal"
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
Line 485: Line 565:
<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 style="width: 257px; height: 194px;" id="Grafik 17"
-
src="Freiburg10_Modularization_GOI-Dateien/image009.jpg"
+
src="https://static.igem.org/mediawiki/2010/0/0a/Freiburg10_Microscopy_phasecontrast_2_GOI.png"
-
alt="Beschreibung: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"
+
alt=""></p>
-
height="194" width="257"></p>
+
</td>
</td>
</tr>
</tr>
<tr>
<tr>
-
<td colspan="2"
+
<td colspan="2" style="padding: 0cm 5.4pt; width: 481.1pt;"
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
+
valign="top" width="641">
valign="top" width="641">
<p class="MsoCaption" style="text-indent: 0cm;"><a
<p class="MsoCaption" style="text-indent: 0cm;"><a
Line 506: Line 584:
</tbody>
</tbody>
</table>
</table>
 +
</div>
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
<h4><a name="_Toc275800685"></a><a name="_Toc275797958"><span
<h4><a name="_Toc275800685"></a><a name="_Toc275797958"><span
lang="EN-US">Analysis
lang="EN-US">Analysis
of Target Cells by Flow Cytometry demonstrates GOI Expression</span></a></h4>
of Target Cells by Flow Cytometry demonstrates GOI Expression</span></a></h4>
-
<p class="MsoNormal"><span lang="EN-US">Characterizing the function of
+
<p class="MsoNormal"><span lang="EN-US">Characterizing
 +
the function of
the hGH
the hGH
-
terminator, the <i>beta-globin</i> intron and the complete plasmid,
+
terminator, the <i>beta-globin</i> intron and the complete
 +
plasmid,
several
several
approaches were conducted followed by analysis via flow cytometry. </span></p>
approaches were conducted followed by analysis via flow cytometry. </span></p>
Line 518: Line 599:
lang="EN-US">Influence
lang="EN-US">Influence
of hGH terminator BioBrick on GOI Expression</span></a></h5>
of hGH terminator BioBrick on GOI Expression</span></a></h5>
-
<p class="MsoNormal"><span lang="EN-US">The iGEM team Freiburg provides
+
<p class="MsoNormal"><span lang="EN-US">The
 +
iGEM team Freiburg provides
the hGH
the hGH
plolyadenylation sequence within the ‘Virus Construction Kit’ due to
plolyadenylation sequence within the ‘Virus Construction Kit’ due to
Line 524: Line 606:
that almost every eukaryotic mRNA is processed at their 3´ and 5´end
that almost every eukaryotic mRNA is processed at their 3´ and 5´end
except for
except for
-
histone mRNAs </span><span lang="EN-US">(Millevoi et al. 2006)</span><span
+
histone mRNAs </span><span lang="EN-US">(Millevoi
-
lang="EN-US">. Pre-mRNAs contain
+
et al. 2006)</span><span lang="EN-US">. Pre-mRNAs
 +
contain
two canonical conserved sequences. First, the polyadenylation signal
two canonical conserved sequences. First, the polyadenylation signal
“AATAAA”
“AATAAA”
Line 536: Line 619:
transcript immediately after a CA-nucleotide therefore defining the
transcript immediately after a CA-nucleotide therefore defining the
cleavage
cleavage
-
site </span><span lang="EN-US">(Danckwardt et al. 2008)</span><span
+
site </span><span lang="EN-US">(Danckwardt et al.
-
style="font-size: 12pt; line-height: 200%;" lang="EN-US">. </span><span
+
2008)</span><span style="font-size: 12pt; line-height: 200%;"
-
lang="EN-US">Recombinant vectorplasmids
+
lang="EN-US">. </span><span lang="EN-US">Recombinant
 +
vectorplasmids
were engineered containing the inverted terminal repeats (ITRs), a
were engineered containing the inverted terminal repeats (ITRs), a
strong
strong
-
eukaryotic promoter (CMV promoter: BBa_K404102) and mVenus as gene of
+
eukaryotic promoter (CMV promoter: <a href = "http://partsregistry.org/Part:BBa_K404102" target="blank" > BBa_K404102</a>) and mVenus as gene of
interest
interest
with and without the hGH terminator signal. Transduction of HT1080
with and without the hGH terminator signal. Transduction of HT1080
Line 547: Line 631:
volume of viral particles containing the vectorplasmids and measuring
volume of viral particles containing the vectorplasmids and measuring
mVenus
mVenus
-
expression 24-hours post infection by flow cytometry demonstrated that
+
expression 24 hours post infection by flow cytometry demonstrated that
transgene expression of the constructs lacking the hGH termination
transgene expression of the constructs lacking the hGH termination
signal is
signal is
-
significantly reduced as shown in </span><span lang="EN-US">Figure 7</span><span
+
significantly reduced as shown in </span><span lang="EN-US">Figure
-
lang="EN-US"> and </span><span lang="EN-US">Figure 8</span><span
+
7</span><span lang="EN-US"> and </span><span lang="EN-US">Figure 8</span><span
-
lang="EN-US"> confirming the expected results
+
lang="EN-US">
 +
confirming the expected results
that hGH is essential for mRNA processing. The iGEM team
that hGH is essential for mRNA processing. The iGEM team
Freiburg_Bioware 2010
Freiburg_Bioware 2010
Line 559: Line 644:
Construction Kit for optimal gene expression.</span></p>
Construction Kit for optimal gene expression.</span></p>
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; border-collapse: collapse;" border="1"
+
style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;"
-
cellpadding="0" cellspacing="0">
+
border="0" cellpadding="0" cellspacing="0">
<tbody>
<tbody>
<tr>
<tr>
<td
<td
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"
+
style="padding: 0cm 5.4pt; vertical-align: top; width: 481.1pt;">
-
valign="top" width="641">
+
<p class="MsoNormal"
<p class="MsoNormal"
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
Line 571: Line 655:
<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-Dateien/image010.gif"
+
id="Grafik 30"
 +
src="https://static.igem.org/mediawiki/2010/c/c7/Freiburg10_FACS_woHGH.png"
height="408" width="629"></p>
height="408" width="629"></p>
</td>
</td>
</tr>
</tr>
<tr>
<tr>
-
<td
+
<td style="padding: 0cm 5.4pt; width: 481.1pt;" valign="top"
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
+
width="641">
-
valign="top" width="641">
+
<p class="MsoNormal"
<p class="MsoNormal"
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
Line 585: Line 669:
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"
id="Grafik 2049"
-
src="Freiburg10_Modularization_GOI-Dateien/image011.gif" height="410"
+
src="https://static.igem.org/mediawiki/2010/7/70/Freiburg10_FACS_withHGH.png"
-
width="635"></p>
+
height="410" width="635"></p>
</td>
</td>
</tr>
</tr>
<tr>
<tr>
-
<td
+
<td style="padding: 0cm 5.4pt; width: 481.1pt;" valign="top"
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
+
width="641">
-
valign="top" width="641">
+
<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
Line 598: Line 681:
lang="EN-US">8</span></b><b><span lang="EN-US">:</span></b><span
lang="EN-US">8</span></b><b><span lang="EN-US">:</span></b><span
lang="EN-US"> <b>Flow cytometry analysis of vectorplasmids with and
lang="EN-US"> <b>Flow cytometry analysis of vectorplasmids with and
-
without hGH terminator.</b> A: Gating non transduced cells (control);
+
without hGH terminator.</b> A: Gating non transduced cells
 +
(control);
subcellular debris and clumps can be distinguished from single cells by
subcellular debris and clumps can be distinguished from single cells by
size, estimated forward scatter (FS Lin) and granularity, estimated
size, estimated forward scatter (FS Lin) and granularity, estimated
Line 605: Line 689:
cells fell within the positive region (R5). C: Gating transduced cells
cells fell within the positive region (R5). C: Gating transduced cells
(R2 </span><span style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;;"
(R2 </span><span style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;;"
-
lang="EN-US">≙</span><span lang="EN-US">R14) (used plasmids for
+
lang="EN-US">≙</span><span lang="EN-US">R14)
 +
(used plasmids for
transfection: GOI: pSB1C3_lITR_CMV_beta-globin intron_mVenus_rITR
transfection: GOI: pSB1C3_lITR_CMV_beta-globin intron_mVenus_rITR
-
(BBa_K404127), pHelper, pRC). D: Transduced cells plotted against
+
(<a href = "http://partsregistry.org/Part:BBa_K404127" target="blank" > BBa_K404127</a>), pHelper, pRC). D: Transduced cells plotted against
mVenus, R10 comprises transduced cells by detecting mVenus expression.
mVenus, R10 comprises transduced cells by detecting mVenus expression.
E: Overlay of non-transduced (red) and transduced (green) cells applied
E: Overlay of non-transduced (red) and transduced (green) cells applied
Line 613: Line 698:
Non-transduced cells applied against mVenus. H: Gating transduced cells
Non-transduced cells applied against mVenus. H: Gating transduced cells
(R2 </span><span style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;;"
(R2 </span><span style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;;"
-
lang="EN-US">≙</span><span lang="EN-US">R14) (used plasmids for
+
lang="EN-US">≙</span><span lang="EN-US">R14)
 +
(used plasmids for
transfection: GOI: reassembled
transfection: GOI: reassembled
-
pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (BBa_K404119), pHelper,
+
pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (<a href = "http://partsregistry.org/Part:BBa_K404119" target="blank" > BBa_K404119</a>), pHelper,
pRC). I: Transduced cells applied against mVenus, R10 comprised
pRC). I: Transduced cells applied against mVenus, R10 comprised
transduced cells, by detecting mVenus expression. J: Overlay of
transduced cells, by detecting mVenus expression. J: Overlay of
Line 625: Line 711:
</table>
</table>
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
 +
<div align="center">
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; border-collapse: collapse;" border="1"
+
style="border: medium none ; border-collapse: collapse;" border="0"
cellpadding="0" cellspacing="0">
cellpadding="0" cellspacing="0">
<tbody>
<tbody>
-
<tr>
+
<tr style="height: 319.2pt;">
-
<td
+
<td style="padding: 0cm 5.4pt; width: 351.1pt; height: 319.2pt;"
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"
+
valign="top" width="468">
-
valign="top" width="641">
+
<p class="MsoNormal"
<p class="MsoNormal"
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
lang="EN-US">&nbsp;</span></p>
lang="EN-US">&nbsp;</span></p>
<p class="MsoNormal"
<p class="MsoNormal"
-
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><span
+
style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
-
lang="EN-US"> </span><img id="Diagramm 3"
+
align="center"><img id="Diagramm 3"
-
src="Freiburg10_Modularization_GOI-Dateien/image012.gif" height="355"
+
src="https://static.igem.org/mediawiki/2010/9/94/Freiburg10_Diagram_hGH.png"
-
width="473"></p>
+
height="355" width="473"></p>
<p class="MsoCaption" style="text-indent: 0cm;"><a
<p class="MsoCaption" style="text-indent: 0cm;"><a
name="_Ref275784545"><span lang="EN-US">Figure </span></a><span
name="_Ref275784545"><span lang="EN-US">Figure </span></a><span
-
lang="EN-US">9</span><span lang="EN-US">: Flow cytometry analysis of
+
lang="EN-US">9</span><span lang="EN-US">:
 +
Flow cytometry analysis of
vectorplasmids with and without hGH terminator.</span><span
vectorplasmids with and without hGH terminator.</span><span
-
style="font-weight: normal;" lang="EN-US"> YFP expression of viral
+
style="font-weight: normal;" lang="EN-US"> YFP
-
genomes was determined by flow cytomery after 24-hour post infection.
+
expression of viral
 +
genomes was determined by flow cytomery 24 hour post infection.
Results demonstrate that mVenus expression of vectorplasmids lacking
Results demonstrate that mVenus expression of vectorplasmids lacking
the hGH terminator is reduced significantly proving that the
the hGH terminator is reduced significantly proving that the
Line 656: Line 744:
</tbody>
</tbody>
</table>
</table>
 +
</div>
<p class="MsoCaption"><span lang="EN-US">&nbsp;</span></p>
<p class="MsoCaption"><span lang="EN-US">&nbsp;</span></p>
<h5><a name="_Toc275800687"></a><a name="_Toc275797960"><span
<h5><a name="_Toc275800687"></a><a name="_Toc275797960"><span
lang="EN-US">Influence
lang="EN-US">Influence
of <i>Beta-globin</i> intron Biobrick on GOI Expression</span></a></h5>
of <i>Beta-globin</i> intron Biobrick on GOI Expression</span></a></h5>
-
<p class="MsoNormal"><span lang="EN-US">Providing an element assumed to
+
<p class="MsoNormal"><span lang="EN-US">Providing
 +
an element assumed to
be an
be an
-
enhancer of transgene expression </span><span lang="EN-US">(Nott et
+
enhancer of transgene expression </span><span lang="EN-US">(Nott
-
al. 2003)</span><span lang="EN-US">, the iGEM team Freiburg tested
+
et
-
a beta-globin intron derived from the human <i>beta globin</i> gene
+
al. 2003)</span><span lang="EN-US">, the iGEM team
 +
Freiburg tested
 +
a beta-globin intron derived from the human <i>beta globin</i>
 +
gene
which can
which can
be fused upstream of the desired gene of interest. The beta-globin
be fused upstream of the desired gene of interest. The beta-globin
Line 670: Line 763:
BioBrick consists of a partial chimeric CMV promoter followed by the
BioBrick consists of a partial chimeric CMV promoter followed by the
intron II
intron II
-
of the <i>beta-globin</i> gene. The 3´end of the intron is fused to
+
of the <i>beta-globin</i> gene. The 3´end of the intron is
 +
fused to
the first 25
the first 25
-
bases of human <i>beta globin</i> gene exon 3. The <i>beta globin</i>
+
bases of human <i>beta globin</i> gene exon 3. The <i>beta
 +
globin</i>
intron
intron
BioBrick is assumed to enhance eukaryotic gene expression </span><span
BioBrick is assumed to enhance eukaryotic gene expression </span><span
Line 678: Line 773:
was conducted as
was conducted as
described for the hGH terminator experiment (see above). As shown in </span><span
described for the hGH terminator experiment (see above). As shown in </span><span
-
lang="EN-US">Figure 9</span><span lang="EN-US"> and </span><span
+
lang="EN-US">Figure 9</span><span lang="EN-US">
-
lang="EN-US">Figure 10</span><span lang="EN-US"> the vectorplasmid
+
and </span><span lang="EN-US">Figure 10</span><span lang="EN-US"> the
 +
vectorplasmid
missing the <i>beta-globin</i>
missing the <i>beta-globin</i>
intron showed a negligible difference in mVenus expression compared to
intron showed a negligible difference in mVenus expression compared to
viral
viral
-
genomes containing the <i>beta-globin</i> intron. Considering these
+
genomes containing the <i>beta-globin</i> intron.
 +
Considering these
results and
results and
taking into account that a constant volume of viral particles has been
taking into account that a constant volume of viral particles has been
Line 691: Line 788:
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 </span><span lang="EN-US">(Dong
decreases with increasing sizes of the insert </span><span lang="EN-US">(Dong
-
et al. 1996)</span><span lang="EN-US">, the iGEM team
+
et al. 1996)</span><span lang="EN-US">, the iGEM
-
Freiburg_Bioware suggests using the <i>beta-globin </i>intron in
+
team
 +
Freiburg_Bioware suggests using the <i>beta-globin </i>intron
 +
in
dependence on
dependence on
the size of your transgene.</span></p>
the size of your transgene.</span></p>
 +
<div align="center">
<table class="MsoTableGrid"
<table class="MsoTableGrid"
style="border: medium none ; width: 490.75pt; border-collapse: collapse;"
style="border: medium none ; width: 490.75pt; border-collapse: collapse;"
-
border="1" cellpadding="0" cellspacing="0" width="654">
+
border="0" cellpadding="0" cellspacing="0" width="654">
<tbody>
<tbody>
<tr style="height: 2.5pt;">
<tr style="height: 2.5pt;">
-
<td
+
<td style="padding: 0cm 5.4pt; width: 490.75pt; height: 2.5pt;"
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 490.75pt; height: 2.5pt;"
+
valign="top" width="654">
valign="top" width="654">
<p class="MsoNormal"
<p class="MsoNormal"
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
-
lang="EN-US">Vectorplasmid lacking <i>beta-globin</i> intron</span></b></p>
+
lang="EN-US">Vectorplasmid lacking <i>beta-globin</i>
 +
intron</span></b></p>
<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 55" src="Freiburg10_Modularization_GOI-Dateien/image013.gif"
+
id="Grafik 55"
 +
src="https://static.igem.org/mediawiki/2010/7/7c/Freiburg10_FACS_betaglobin.png"
height="412" width="640"></p>
height="412" width="640"></p>
</td>
</td>
</tr>
</tr>
<tr style="height: 106.5pt;">
<tr style="height: 106.5pt;">
-
<td
+
<td style="padding: 0cm 5.4pt; width: 490.75pt; height: 106.5pt;"
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 490.75pt; height: 106.5pt;"
+
valign="top" width="654">
valign="top" width="654">
<p class="MsoNormal"
<p class="MsoNormal"
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
-
lang="EN-US">Vectorplasmid containing <i>beta-globin</i> intron</span></b></p>
+
lang="EN-US">Vectorplasmid containing <i>beta-globin</i>
 +
intron</span></b></p>
<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 63" src="Freiburg10_Modularization_GOI-Dateien/image014.gif"
+
id="Grafik 63"
 +
src="https://static.igem.org/mediawiki/2010/8/83/Freiburg10_FACS_withbetaglobin.png"
height="410" width="635"></p>
height="410" width="635"></p>
</td>
</td>
</tr>
</tr>
<tr style="height: 106.5pt;">
<tr style="height: 106.5pt;">
-
<td
+
<td style="padding: 0cm 5.4pt; width: 490.75pt; height: 106.5pt;"
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 490.75pt; height: 106.5pt;"
+
valign="top" width="654">
valign="top" width="654">
<p class="MsoCaption" style="text-indent: 0cm;"><a
<p class="MsoCaption" style="text-indent: 0cm;"><a
name="_Ref275784803"><span lang="EN-US">Figure </span></a><span
name="_Ref275784803"><span lang="EN-US">Figure </span></a><span
-
lang="EN-US">10</span><span lang="EN-US">: Flow cytometry analysis of
+
lang="EN-US">10</span><span lang="EN-US">:
-
vectorplasmids with and without <i>beta-globin</i> intron. A</span><span
+
Flow cytometry analysis of
-
style="font-weight: normal;" lang="EN-US">: Gating non transduced
+
vectorplasmids with and without <i>beta-globin</i> intron.
 +
A</span><span style="font-weight: normal;" lang="EN-US">:
 +
Gating non transduced
cells (control); subcellular debris and clumps can be distinguished
cells (control); subcellular debris and clumps can be distinguished
from single cells by size, estimated forward scatter (FS Lin) and
from single cells by size, estimated forward scatter (FS Lin) and
granularity, estimated side scatter (SS Lin) </span><span lang="EN-US">B</span><span
granularity, estimated side scatter (SS Lin) </span><span lang="EN-US">B</span><span
-
style="font-weight: normal;" lang="EN-US">: Non transduced cells
+
style="font-weight: normal;" lang="EN-US">: Non
 +
transduced cells
applied against mVenus (Analytical gate was set such that 1% or fewer
applied against mVenus (Analytical gate was set such that 1% or fewer
of negative control cells fell within the positive region (R5). </span><span
of negative control cells fell within the positive region (R5). </span><span
Line 745: Line 849:
lang="EN-US">≙</span><span style="font-weight: normal;" lang="EN-US">R14)
lang="EN-US">≙</span><span style="font-weight: normal;" lang="EN-US">R14)
(used plasmids for transfection: GOI: </span><span lang="EN-US">pSB1C3_lITR_CMV_mVenus_hGH_rITR
(used plasmids for transfection: GOI: </span><span lang="EN-US">pSB1C3_lITR_CMV_mVenus_hGH_rITR
-
(BBa_K404128)</span><span style="font-weight: normal;" lang="EN-US">,
+
(<a href = "http://partsregistry.org/Part:BBa_K404128" target="blank" > BBa_K404128</a>)</span><span style="font-weight: normal;" lang="EN-US">,
pHelper, pRC). </span><span lang="EN-US">D</span><span
pHelper, pRC). </span><span lang="EN-US">D</span><span
-
style="font-weight: normal;" lang="EN-US">: Transduced cells plotted
+
style="font-weight: normal;" lang="EN-US">:
 +
Transduced cells plotted
against mVenus, R10 comprised transduced cells, by detecting mVenus
against mVenus, R10 comprised transduced cells, by detecting mVenus
expression </span><span lang="EN-US">E</span><span
expression </span><span lang="EN-US">E</span><span
-
style="font-weight: normal;" lang="EN-US">: Overlay of non-transduced
+
style="font-weight: normal;" lang="EN-US">: Overlay
 +
of non-transduced
(red) and transduced (green) cells applied against mVenus </span><span
(red) and transduced (green) cells applied against mVenus </span><span
lang="EN-US">F</span><span style="font-weight: normal;" lang="EN-US">:
lang="EN-US">F</span><span style="font-weight: normal;" lang="EN-US">:
Gating non-transduced cells (control). </span><span lang="EN-US">G</span><span
Gating non-transduced cells (control). </span><span lang="EN-US">G</span><span
-
style="font-weight: normal;" lang="EN-US">: Non-transduced cells
+
style="font-weight: normal;" lang="EN-US">:
 +
Non-transduced cells
applied against mVenus (R5).</span><span lang="EN-US">H</span><span
applied against mVenus (R5).</span><span lang="EN-US">H</span><span
-
style="font-weight: normal;" lang="EN-US">: Gating transduced cells
+
style="font-weight: normal;" lang="EN-US">: Gating
 +
transduced cells
(R2 </span><span
(R2 </span><span
style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;"
style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;"
lang="EN-US">≙</span><span style="font-weight: normal;" lang="EN-US">R14)
lang="EN-US">≙</span><span style="font-weight: normal;" lang="EN-US">R14)
(used plasmids for transfection: GOI: reassembled </span><span
(used plasmids for transfection: GOI: reassembled </span><span
-
lang="EN-US">pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (BBa_K404119)</span><span
+
lang="EN-US">pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
-
style="font-weight: normal;" lang="EN-US">, pHelper, pRC). </span><span
+
(<a href = "http://partsregistry.org/Part:BBa_K404119" target="blank" > BBa_K404119</a>)</span><span style="font-weight: normal;" lang="EN-US">,
-
lang="EN-US">I</span><span style="font-weight: normal;" lang="EN-US">:
+
pHelper, pRC). </span><span lang="EN-US">I</span><span
 +
style="font-weight: normal;" lang="EN-US">:
Transduced cells applied against mVenus, R10 comprised transduced
Transduced cells applied against mVenus, R10 comprised transduced
cells, by detecting mVenus expression. </span><span lang="EN-US">J</span><span
cells, by detecting mVenus expression. </span><span lang="EN-US">J</span><span
-
style="font-weight: normal;" lang="EN-US">: Overlay of non-transduced
+
style="font-weight: normal;" lang="EN-US">: Overlay
 +
of non-transduced
(red) and transduced (green) cells applied against mVenus.</span></p>
(red) and transduced (green) cells applied against mVenus.</span></p>
</td>
</td>
Line 772: Line 882:
</tbody>
</tbody>
</table>
</table>
 +
</div>
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
 +
<div align="center">
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; width: 490.75pt; border-collapse: collapse;"
+
style="border: medium none ; width: 306.65pt; border-collapse: collapse;"
-
border="1" cellpadding="0" cellspacing="0" width="654">
+
border="0" cellpadding="0" cellspacing="0" width="409">
<tbody>
<tbody>
-
<tr style="height: 90.2pt;">
+
<tr style="height: 87.3pt;">
-
<td
+
<td style="padding: 0cm 5.4pt; width: 306.65pt; height: 87.3pt;"
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 490.75pt; height: 90.2pt;"
+
valign="top" width="409">
-
valign="top" width="654">
+
<p class="MsoNormal"
<p class="MsoNormal"
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"><img id="Diagramm 57"
align="center"><img id="Diagramm 57"
-
src="Freiburg10_Modularization_GOI-Dateien/image015.gif" height="332"
+
src="https://static.igem.org/mediawiki/2010/2/26/Freiburg10_Diagram_betaglobin.png.png"
-
width="450"></p>
+
height="332" width="450"></p>
<p class="MsoCaption" style="text-indent: 0cm;"><a
<p class="MsoCaption" style="text-indent: 0cm;"><a
name="_Ref275784805"><span lang="EN-US">Figure </span></a><span
name="_Ref275784805"><span lang="EN-US">Figure </span></a><span
-
lang="EN-US">11</span><span lang="EN-US">: Flow cytometry analysis of
+
lang="EN-US">11</span><span lang="EN-US">:
 +
Flow cytometry analysis of
vectorplasmids with and without <i>beta-globin</i> intron.</span><span
vectorplasmids with and without <i>beta-globin</i> intron.</span><span
-
style="font-weight: normal;" lang="EN-US"> 48-hours post transfection,
+
style="font-weight: normal;" lang="EN-US"> 48 hours
 +
post transfection,
viral particles were harvested by freeze-thaw lysis and centrifugation
viral particles were harvested by freeze-thaw lysis and centrifugation
followed by HT1080 transduction. YFP expression of vectorplasmids was
followed by HT1080 transduction. YFP expression of vectorplasmids was
-
determined by flow cytometry 24-hours post infection. The vectorplasmid
+
determined by flow cytometry 24 hours post infection. The vectorplasmid
missing the beta-globin intron showed a negligible difference in mVenus
missing the beta-globin intron showed a negligible difference in mVenus
expression compared to viral plasmid containing the beta-globin intron.</span></p>
expression compared to viral plasmid containing the beta-globin intron.</span></p>
Line 800: Line 913:
</tbody>
</tbody>
</table>
</table>
 +
</div>
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
<h5><a name="_Toc275800688"></a><a name="_Toc275797961"><span
<h5><a name="_Toc275800688"></a><a name="_Toc275797961"><span
Line 805: Line 919:
of the Full Assembled Vectorplasmid Demonstrated by GOI Expression</span></a><span
of the Full Assembled Vectorplasmid Demonstrated by GOI Expression</span></a><span
lang="EN-US"> </span></h5>
lang="EN-US"> </span></h5>
-
<p class="MsoNormal"><span lang="EN-US">After assembly of plasmids
+
Producing recombinant virus particles for therapeutical applications
-
containing all
+
is, besides specific cell targeting, purification and quantification
-
required elements (see </span><span lang="EN-US">Figure 1</span><span
+
assays of AAV-2, one intention of the Virus Construction Kit provided
-
lang="EN-US">),
+
by the iGEM team Freiburg_Bioware 2010. For obtaining a modular
-
functionality was tested in cell culture. AAV-293 cells stably
+
toolkit, the complex biological system of the Adeno-associated virus
-
expressing E1A
+
serotype 2 was examined by an exhaustive literature search.
-
and E1B proteins were transfected with three plasmids (pHelper, pRC,
+
Subsequently, the essential components for AAV-2 particle production
-
pGOI).
+
were extracted and redesigned to match the iGEM standard.<br>
-
Virus particles were harvested 72-hours post-transfection and the tumor
+
The provided tripartite system is independent of a superinfection&nbsp;
-
cell
+
of Adeno- or herpes simplex viruses since the genes encoding the
-
line HT1080 was transduced with the recombinant viral vectors
+
required helper-proteins are co-transfected. Inside the eukaryotic host
-
encapsidating the
+
cell, the DNA sequence containing the inverted terminal repeats (ITRs)
-
gene of interest mVenus (BBa_I757008).</span></p>
+
is extracted and later encapsidated into the preformed capsids after
-
<p class="MsoNormal"><span lang="EN-US">The iGEM team Freiburg_Bioware
+
production of single-stranded DNA. Consequently, this plasmid is known
-
2010
+
as the vector plasmid (pGOI). Promoter, beta-globin intron and the hGH
-
compared the standard-plasmid containing a subcloned mVenus
+
terminator signal are flanked by the ITRs (ITRs, <a href = "http://partsregistry.org/Part:BBa_K404100" target="blank" > BBa_K404100</a> and
-
(pAAV_mVenus,
+
<a href = "http://partsregistry.org/Part:BBa_K404101" target="blank" > BBa_K404101</a>) and regulate transgene expression. The vector plasmid
-
derived from the Stratagene system) with the assembled plasmid
+
containing the desired gene of interest is cotransfected with the
-
pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
+
RepCap plasmid (<a href = "http://partsregistry.org/Part:BBa_K404001" target="blank" > BBa_K404001</a>, <a href = "http://partsregistry.org/Part:BBa_K404102" target="blank" > BBa_K404102</a> or <a href = "http://partsregistry.org/Part:BBa_K404103" target="blank" > BBa_K404103</a>) and the
-
(pSB1C3_mVenus: BBa_K404119). Fluorescence expression data obtained by
+
pHelper plasmid. To obtain the fully assembled vector plasmid, several
-
flow
+
assembly steps have to be performed. &nbsp;<br>
-
cytometry analysis are shown in </span><span lang="EN-US">Figure 11</span><span
+
After assembly of plasmids containing all required elements, vector
-
lang="EN-US"> and </span><span lang="EN-US">Figure 12</span><span
+
plasmid functionality was confirmed in cell culture. AAV-293 cells
-
lang="EN-US">.
+
stably expressing the E1A and E1B proteins were transfected with three
-
Comparing mVenus expression of the standard plasmid and the modified,
+
plasmids (pHelper, pRC, pGOI). Virus particles were harvested 72 hours
-
assembled
+
post transfection and the tumor cell line HT1080 was transduced with
-
plasmid reveals that biological functionality of the reassembled
+
the recombinant viral vectors encapsidating the gene of interest mVenus
-
plasmid was
+
(<a href = "http://partsregistry.org/Part:BBa_I757008" target="blank" > BBa_I757008</a>).<br>
-
confirmed. </span></p>
+
In the beginning, the iGEM team Freiburg_Bioware 2010 used a commercial
 +
vector plasmid, pAAV-MCS (Stratagene), to determine whether virus
 +
particle production by AAV-293 cells could be achieved. iGEM RFC 25
 +
restriction enzyme sites were introduced and the fluorescent protein
 +
mVenus was subcloned into this plasmid. Subsequently, this plasmid was
 +
used as a reference and compared to the assembled vector plasmid
 +
pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (abbreviated pSB1C3_mVenus,
 +
<a href = "http://partsregistry.org/Part:BBa_K404119" target="blank" > BBa_K404119</a>). Fluorescence expression data obtained by flow cytometry
 +
analysis are shown in Figure 11 and Figure 12. Comparing mVenus
 +
expression of the reference plasmid and the pSB1C3_mVenus plasmid
 +
revealed that biological functionality of the reassembled plasmid was
 +
preserved. <br>
 +
<br>
 +
<div align="center">
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; border-collapse: collapse;" border="1"
+
style="border: medium none ; border-collapse: collapse;" border="0"
cellpadding="0" cellspacing="0">
cellpadding="0" cellspacing="0">
<tbody>
<tbody>
<tr>
<tr>
-
<td
+
<td style="padding: 0cm 5.4pt; width: 481.1pt;" valign="top"
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"
+
width="641">
-
valign="top" width="641">
+
<p class="MsoNormal"
<p class="MsoNormal"
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>pSB1C3_mVenus
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>pSB1C3_mVenus
-
(BBa_K404119)</b></p>
+
(<a href = "http://partsregistry.org/Part:BBa_K404119" target="blank" > BBa_K404119</a>)</b></p>
<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 2068"
id="Grafik 2068"
-
src="Freiburg10_Modularization_GOI-Dateien/image016.gif" height="409"
+
src="https://static.igem.org/mediawiki/2010/9/9e/Freiburg10_FACS_FULL_pSB1c3_mVenus.png"
-
width="634"></p>
+
height="409" width="634"></p>
</td>
</td>
</tr>
</tr>
<tr>
<tr>
-
<td
+
<td style="padding: 0cm 5.4pt; width: 481.1pt;" valign="top"
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
+
width="641">
-
valign="top" width="641">
+
<p class="MsoNormal"
<p class="MsoNormal"
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
-
lang="EN-US">pAAV_mVenus (Stratagene)</span></b></p>
+
lang="EN-US">pAAV_mVenus (reference)<br>
 +
</span></b></p>
<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 89" src="Freiburg10_Modularization_GOI-Dateien/image017.gif"
+
id="Grafik 89"
 +
src="https://static.igem.org/mediawiki/2010/a/ad/Freiburg10_FACS_FULL_pAAV_mVenus.png"
height="407" width="630"></p>
height="407" width="630"></p>
</td>
</td>
</tr>
</tr>
<tr>
<tr>
-
<td
+
<td style="padding: 0cm 5.4pt; width: 481.1pt;" valign="top"
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
+
width="641">
-
valign="top" width="641">
+
<p class="MsoCaption" style="text-indent: 0cm;"><a
<p class="MsoCaption" style="text-indent: 0cm;"><a
name="_Ref275784576"><span lang="EN-US">Figure </span></a><span
name="_Ref275784576"><span lang="EN-US">Figure </span></a><span
lang="EN-US">12</span><span lang="EN-US">: Flow cytometry analysis of
lang="EN-US">12</span><span lang="EN-US">: Flow cytometry analysis of
-
reassembled vectorplasmid (BBa_K404119) compared to standard plasmid
+
fluorescent protein expression in transduced HT1080 cells. For viral
-
provided by Stratagene. A</span><span style="font-weight: normal;"
+
particle production, AAV-293 cells were transfected with the
-
lang="EN-US">: Gating non transduced cells (control); subcellular
+
reassembled vector plasmid (<a href = "http://partsregistry.org/Part:BBa_K404119" target="blank" > BBa_K404119</a>) or the reference plasmid,
-
debris and clumps can be distinguished from single cells by size,
+
respectively. A: Gating non transduced cells (control); subcellular
-
estimated forward scatter (FS Lin) and granularity, estimated side
+
debris and cellular aggreates can be distinguished from single cells by
-
scatter (SS Lin) B: Non transduced cells plotted against mVenus
+
size, estimated forward scatter (FS Lin) and granularity, estimated
-
(Analytical gate was set such that 1% or fewer of negative control
+
side scatter (SS Lin) B: Non-transduced cells plotted against cells
-
cells fell within the positive region (R5).C: Gating transduced cells
+
expressing mVenus (Analytical gate was set such that 1% or fewer of
-
(R2 </span><span
+
negative control cells fell within the positive region (R5) C: Gating
-
style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;"
+
transduced cells (R2 ≙ R14) (plasmids used for transfection: pGOI:
-
lang="EN-US"></span><span style="font-weight: normal;" lang="EN-US">R14)
+
pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (pSB1C3_mVenus:
-
(used plasmids for transfection: pGOI: </span><span lang="EN-US">pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
+
<a href = "http://partsregistry.org/Part:BBa_K404119" target="blank" > BBa_K404119</a>), pHelper, pRC. D: Transduced cells plotted against cells
-
(pSB1C3_mVenus: </span><span lang="EN-US">BBa_K404119</span><span
+
expressing mVenus. R10 comprises transduced cells detected by mVenus
-
style="color: rgb(0, 176, 80); font-weight: normal;" lang="EN-US">)</span><span
+
fluorescence. E: Overlay of non-transduced (red) and transduced
-
style="font-weight: normal;" lang="EN-US">, pHelper, pRC. </span><span
+
(green). F: Gating non-transduced cells (control). G: Non-transduced
-
lang="EN-US">D</span><span style="font-weight: normal;" lang="EN-US">:
+
cells plotted against cells expressing mVenus (R5). H: Gating
-
Transduced cells plotted against mVenus, R10 comprised transduced
+
transduced cells (R14 ≙ R2) (plasmids used for transfection: pGOI:
-
cells, by detecting mVenus expression. </span><span lang="EN-US">E</span><span
+
pAAV_mVenus, pHelper). I: Transduced cells plotted against cells
-
style="font-weight: normal;" lang="EN-US">: Overlay of non-transduced
+
expressing mVenus. R10 comprises transduced cells detected by mVenus
-
(red) and transduced (green). </span><span lang="EN-US">F</span><span
+
fluorescence. J: Overlay of non-transduced (red) and transduced (green)
-
style="font-weight: normal;" lang="EN-US">: Gating non transduced
+
cells plotted against mVenus expression.&nbsp;</span><span
-
cells (control). </span><span lang="EN-US">G</span><span
+
style="font-weight: normal;" lang="EN-US"> </span></p>
-
style="font-weight: normal;" lang="EN-US">: Non-transduced cells
+
-
plotted against mVenus (R5). </span><span lang="EN-US">H</span><span
+
-
style="font-weight: normal;" lang="EN-US">: Gating transduced cells
+
-
(R14 </span><span
+
-
style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;"
+
-
lang="EN-US"></span><span style="font-weight: normal;" lang="EN-US">R2)
+
-
(used plasmids for transfection: pGOI: pAAV_mVenus, pHelper). </span><span
+
-
lang="EN-US">I</span><span style="font-weight: normal;" lang="EN-US">:
+
-
Transduced cells plotted against mVenus, R10 comprised transduced
+
-
cells, by detecting mVenus expression.</span><span lang="EN-US"> J</span><span
+
-
style="font-weight: normal;" lang="EN-US">: Overlay of non-transduced
+
-
(red) and transduced (green) cells plotted against mVenus expression. </span></p>
+
</td>
</td>
</tr>
</tr>
</tbody>
</tbody>
</table>
</table>
 +
</div>
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
 +
<div align="center">
<table class="MsoTableGrid"
<table class="MsoTableGrid"
-
style="border: medium none ; border-collapse: collapse;" border="1"
+
style="border: medium none ; border-collapse: collapse;" border="0"
cellpadding="0" cellspacing="0">
cellpadding="0" cellspacing="0">
<tbody>
<tbody>
<tr>
<tr>
-
<td
+
<td style="padding: 0cm 5.4pt; width: 481.1pt;" valign="top"
-
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"
+
width="641">
-
valign="top" width="641">
+
<p class="MsoNormal"
<p class="MsoNormal"
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
Line 926: Line 1,041:
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"><img id="Diagramm 90"
align="center"><img id="Diagramm 90"
-
src="Freiburg10_Modularization_GOI-Dateien/image018.gif" height="396"
+
src="https://static.igem.org/mediawiki/2010/9/90/Freiburg10_Diagram_FULL_GOI.png"
-
width="541"></p>
+
height="396" width="541"></p>
<p class="MsoCaption" style="text-indent: 0cm;"><a
<p class="MsoCaption" style="text-indent: 0cm;"><a
name="_Ref275784852"><span lang="EN-US">Figure </span></a><span
name="_Ref275784852"><span lang="EN-US">Figure </span></a><span
-
lang="EN-US">13</span><span lang="EN-US">: Flow cytometry analysis of
+
lang="EN-US">13</span><span lang="EN-US">: Quantification of mVenus
-
reassembled vectorplasmid (BBa_K404119) compared to standard plasmid
+
fluorescence by flow cytometry analysis. The data source is identical
-
provided by Stratagene. </span><span style="font-weight: normal;"
+
to Figure 13. Compared to the reference vector plasmid, mVenus
-
lang="EN-US">Fluorescence of the standard plasmid pAAV_mVenus
+
expression in HT1080 cells is enhanced in case of the assembled plasmid
-
(Stratagene) and the recombinant pSB1C3_mVenus (BBa_K404119) construct
+
(pSB1C3_mVenus)&nbsp;</span><span style="font-weight: normal;"
-
was measured. As demonstrated mVenus expression is enhanced in the
+
lang="EN-US"></span></p>
-
assembled plasmid (pSB1C3_mVenus) compared to the standard pAAV_mVenus
+
-
construct.</span></p>
+
</td>
</td>
</tr>
</tr>
</tbody>
</tbody>
</table>
</table>
 +
</div>
<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800689"><span
<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800689"><span
lang="EN-US"><span
lang="EN-US"><span
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"></span></span><span
style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"></span></span><span
lang="EN-US">Conclusion</span></a></h3>
lang="EN-US">Conclusion</span></a></h3>
-
<p class="MsoNormal"><span lang="EN-US">Idea of the modular ‘Virus
+
<p class="MsoNormal"><span lang="EN-US">Idea
 +
of the modular ‘Virus
Construction
Construction
Kit’ is to provide all required elements for producing recombinant,
Kit’ is to provide all required elements for producing recombinant,
Line 972: Line 1,087:
cytosine deaminase. Further details demonstrating efficient tumor
cytosine deaminase. Further details demonstrating efficient tumor
killing,
killing,
-
using prodrug-activating systems, see results page ‘Arming – Killing
+
using prodrug-activating systems, see results page <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results#modularization">Arming</a>. </span></p>
-
the
+
-
tumor’. </span></p>
+
<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800690"><span
<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800690"><span
lang="EN-US"><span
lang="EN-US"><span
Line 984: Line 1,097:
A.E., 2008. 3' end mRNA processing: molecular mechanisms and
A.E., 2008. 3' end mRNA processing: molecular mechanisms and
implications for
implications for
-
health and disease. <i>The EMBO journal</i>, 27(3), 482-98. Available
+
health and disease. <i>The EMBO journal</i>, 27(3),
 +
482-98. Available
at:
at:
http://www.ncbi.nlm.nih.gov/pubmed/18256699.</span></p>
http://www.ncbi.nlm.nih.gov/pubmed/18256699.</span></p>
Line 991: Line 1,105:
lang="EN-US">Dong, J.Y., Fan, P.D. &amp; Frizzell, R.a.,
lang="EN-US">Dong, J.Y., Fan, P.D. &amp; Frizzell, R.a.,
1996. Quantitative analysis of the packaging capacity of recombinant
1996. Quantitative analysis of the packaging capacity of recombinant
-
adeno-associated virus. <i>Human gene therapy</i>, 7(17), 2101-12.
+
adeno-associated virus. <i>Human gene therapy</i>, 7(17),
 +
2101-12.
Available
Available
at: http://www.ncbi.nlm.nih.gov/pubmed/8934224.</span></p>
at: http://www.ncbi.nlm.nih.gov/pubmed/8934224.</span></p>
Line 998: Line 1,113:
lang="EN-US">Millevoi, S. et al., 2006. An interaction
lang="EN-US">Millevoi, S. et al., 2006. An interaction
between U2AF 65 and CF I(m) links the splicing and 3' end processing
between U2AF 65 and CF I(m) links the splicing and 3' end processing
-
machineries. <i>The EMBO journal</i>, 25(20), 4854-64. Available at:
+
machineries. <i>The EMBO journal</i>, 25(20), 4854-64.
 +
Available at:
http://www.ncbi.nlm.nih.gov/pubmed/17024186.</span></p>
http://www.ncbi.nlm.nih.gov/pubmed/17024186.</span></p>
<p style="text-indent: 36pt;"><span
<p style="text-indent: 36pt;"><span
Line 1,007: Line 1,123:
(New York, N.Y.)</i>, 9(5), 607-17. Available at:
(New York, N.Y.)</i>, 9(5), 607-17. Available at:
http://www.ncbi.nlm.nih.gov/pubmed/12702819.</span></p>
http://www.ncbi.nlm.nih.gov/pubmed/12702819.</span></p>
-
<p style="text-indent: 36pt;"><span lang="EN-US">&nbsp;</span></p>
+
<p style="text-indent: 36pt;"><span lang="EN-US">&nbsp;</span></p><br>
 +
 
 +
 
 +
 
 +
<a href = "https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid">Move to Top</a>
 +
 
 +
 
 +
 
 +
<p class="MsoNormal"
 +
style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;"
 +
align="left"><span lang="EN-US">&nbsp;</span></p>
 +
<h2 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275885921"></a><a
 +
name="_Toc275817880"><span lang="EN-US">Overview of RepVP123 plasmid</span></a></h2>
 +
<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275885922"></a><a
 +
name="_Toc275817881"><span lang="EN-US">Modularization: Overview</span></a></h3>
 +
<p class="MsoNormal"
 +
style="text-indent: 0cm; line-height: 150%; page-break-after: avoid;"><span
 +
lang="EN-US">In our terminology the term “RepVP123”
 +
encompasses the
 +
whole AAV2 genome excluding the ITRs. The <i>rep</i> locus
 +
comprises four
 +
proteins related to genome replication while the <i>cap</i>
 +
locus codes for the
 +
proteins VP1, VP2, VP3 and the assembly-associated protein (AAP), which
 +
are
 +
required for viral capsid assembly. Source of the RepVP123 BioBrick
 +
supplied
 +
within iGEM team Freiburg_Bioware 2010 Virus Construction Kit is the
 +
wild-type
 +
AAV2 RepVP123, as provided e. g. in the pAAV vector from Stratagene. In
 +
order
 +
to introduce iGEM standard and additionally enabling the possibility to
 +
modify
 +
the viral capsid via integration of certain motives within the viral
 +
loops 453
 +
and 587 a total of twelve mutations within RepVP123 (see </span><span
 +
lang="EN-US">Figure 1</span><span lang="EN-US">)
 +
and additionally two mutations
 +
within the pSB1C3 backbone were performed either by Site-Directed
 +
Mutagenesis
 +
(SDM) or by ordering and cloning of specifically designed gene
 +
sequences matching
 +
the required demands. Modifying the pSB1C3 led to iGEM team
 +
Freiburg_Bioware’s
 +
variant of this backbone, pSB1C3_001.</span></p>
 +
<table class="MsoTableGrid"
 +
style="border: medium none ; border-collapse: collapse; width: 636px; height: 523px; text-align: left; margin-left: auto; margin-right: auto;"
 +
border="0" cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr>
 +
<td
 +
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 462.5pt;">
 +
<p class="MsoNormal"
 +
style="text-align: center; text-indent: 0cm; line-height: 150%; page-break-after: avoid;"
 +
align="center"><img style="width: 605px; height: 451px;"
 +
id="Picture 17"
 +
src="https://static.igem.org/mediawiki/2010/0/0b/Freiburg10_pAAV_complete_mutations.png"
 +
alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\RepCap_complete_modifications_arrows.jpg"></p>
 +
<p class="MsoCaption" style="text-indent: 0cm;"><a
 +
name="_Ref275820820"><span lang="EN-US">Figure </span></a><span
 +
lang="EN-US">1</span> <span
 +
style="font-size: 8pt; color: windowtext; font-weight: normal;"
 +
lang="EN-US">Mutations implemented into <i>RepVP123</i>
 +
in order to establish both iGEM standard and loop insertion capability.
 +
Green arrows indicate integrated restriction sites, red red arrows
 +
indicate deleted restriction sites. KpnI was deleted first and
 +
reinstated afterwards. (see text).</span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<span
 +
style="font-size: 8pt; line-height: 150%; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
 +
lang="EN-US"><br style="page-break-before: always;" clear="all">
 +
</span>
 +
<p class="MsoNormal" style="text-indent: 0cm; line-height: 150%;"><span
 +
style="font-size: 8pt; line-height: 150%;" lang="EN-US">&nbsp;</span></p>
 +
<table class="MsoNormalTable"
 +
style="border-collapse: collapse; width: 651px; height: 261px; text-align: left; margin-left: auto; margin-right: auto;"
 +
border="0" cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr style="height: 27pt;">
 +
<td
 +
style="border: 1pt solid windowtext; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 27pt;">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">Plasmid
 +
name:</span></p>
 +
</td>
 +
<td
 +
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 27pt;"
 +
width="103">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">Functionality
 +
(determinded in cell culture via transduction and flow cytometry ):</span></p>
 +
</td>
 +
<td
 +
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 27pt;"
 +
width="104">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">4x
 +
mutations (PstI (310), BamHI (859), SalI (1239), PstI (4073))</span></p>
 +
</td>
 +
<td
 +
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 27pt;"
 +
width="57">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;">inserted <i>rep</i>
 +
fragment</span></p>
 +
</td>
 +
<td
 +
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 27pt;"
 +
width="66">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;">inserted<i> cap</i>
 +
fragment</span></p>
 +
</td>
 +
<td
 +
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 27pt;"
 +
width="67">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;">reinstated KpnI</span></p>
 +
</td>
 +
<td
 +
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 27pt;"
 +
width="32">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;">pAAV</span></p>
 +
</td>
 +
<td
 +
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 27pt;"
 +
width="70">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;">pSB1C3_001</span></p>
 +
</td>
 +
<td
 +
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 27pt;"
 +
width="37">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;">HSPG-ko</span></p>
 +
</td>
 +
</tr>
 +
<tr style="height: 15pt;">
 +
<td
 +
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 15pt;"
 +
width="115">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC
 +
(wild-type)</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 15pt;"
 +
width="103">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 15pt;"
 +
width="104">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="57">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="66">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="67">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="32">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="70">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="37">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
</tr>
 +
<tr style="height: 15pt;">
 +
<td
 +
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 15pt;"
 +
width="115">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC_4x
 +
mutant</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 15pt;"
 +
width="103">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 15pt;"
 +
width="104">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="57">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="66">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="67">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="32">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="70">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="37">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
</tr>
 +
<tr style="height: 15pt;">
 +
<td
 +
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 15pt;"
 +
width="115">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC_inserts </span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 15pt;"
 +
width="103">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">no</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 15pt;"
 +
width="104">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="57">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="66">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="67">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="32">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="70">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="37">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
</tr>
 +
<tr style="height: 15pt;">
 +
<td
 +
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 15pt;"
 +
width="115">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC_Cap</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 15pt;"
 +
width="103">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 15pt;"
 +
width="104">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="57">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="66">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="67">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="32">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="70">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="37">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
</tr>
 +
<tr style="height: 15pt;">
 +
<td
 +
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 15pt;"
 +
width="115">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC_RepVP123</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 15pt;"
 +
width="103">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 15pt;"
 +
width="104">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="57">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="66">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="67">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="32">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="70">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 15pt;"
 +
nowrap="nowrap" valign="bottom" width="37">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
</tr>
 +
<tr style="height: 18pt;">
 +
<td
 +
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 18pt;"
 +
width="115">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">pSB1C3_RepVP123_
 +
p5TATAless</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 18pt;"
 +
width="103">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 18pt;"
 +
width="104">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 18pt;"
 +
nowrap="nowrap" valign="bottom" width="57">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 18pt;"
 +
nowrap="nowrap" valign="bottom" width="66">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 18pt;"
 +
nowrap="nowrap" valign="bottom" width="67">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 18pt;"
 +
nowrap="nowrap" valign="bottom" width="32">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 18pt;"
 +
nowrap="nowrap" valign="bottom" width="70">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 18pt;"
 +
nowrap="nowrap" valign="bottom" width="37">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
</tr>
 +
<tr style="height: 27pt;">
 +
<td
 +
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 27pt;"
 +
width="115">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">pSB1C3_RepVP123_
 +
HSPG-ko_p5TATAless</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 27pt;"
 +
width="103">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 27pt;"
 +
width="104">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
 +
style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 27pt;"
 +
nowrap="nowrap" valign="bottom" width="57">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 27pt;"
 +
nowrap="nowrap" valign="bottom" width="66">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 27pt;"
 +
nowrap="nowrap" valign="bottom" width="67">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 27pt;"
 +
nowrap="nowrap" valign="bottom" width="32">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 27pt;"
 +
nowrap="nowrap" valign="bottom" width="70">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
<td
 +
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 27pt;"
 +
nowrap="nowrap" valign="bottom" width="37">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
 +
align="left"><span style="font-size: 10pt; color: black;">x</span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<p class="MsoCaption" style="text-align: left;" align="left"><span
 +
lang="EN-US">Figure </span><span lang="EN-US">2</span> <span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">Table
 +
contains complete overview about all plasmids containing <i>RepVP123</i>
 +
which
 +
were used by iGEM team Freiburg_Bioware 2010.</span></p>
 +
<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275885923"></a><a
 +
name="_Toc275817882"><span lang="EN-US">Modularization: Removing iGEM
 +
restriction
 +
sites and establishing loop insertion capability</span></a></h3>
 +
<h4 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275885924"></a><a
 +
name="_Toc275817883"><span lang="EN-US">Modifications in Rep</span></a></h4>
 +
<table class="MsoTableGrid"
 +
style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;"
 +
border="0" cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr>
 +
<td
 +
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 460.6pt;">
 +
<p class="MsoNormal"
 +
style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
 +
align="center"><img style="width: 576px; height: 191px;" id="Picture 4"
 +
src="https://static.igem.org/mediawiki/2010/b/bc/Freiburg10_rep_synthetic_gene_fragment.png"
 +
alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\Rep_synthesis_marked.jpg"></p>
 +
<p class="MsoCaption"><a name="_Ref275818017"></a><a
 +
name="_Ref275818042"><span lang="EN-US">Figure </span></a><span
 +
lang="EN-US">3</span> <span
 +
style="font-size: 8pt; color: windowtext; font-weight: normal;"
 +
lang="EN-US">Restriction sites within the wild-type <i>rep</i>
 +
gene sequence, which were removed via cloning of synthetized <i>rep </i>gene
 +
fragment into the plasmid. The red box indicates
 +
the region spanned by the synthetic sequence.</span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">Making
 +
the <i>RepVP123</i>
 +
wild-type compatible with the iGEM standards required the removal of
 +
five
 +
restriction sites (see </span><span lang="EN-US">Figure
 +
1</span><span lang="EN-US">).
 +
This was achieved using site-directed mutagenesis for PstI (position
 +
310) and
 +
PstI (4073). The remaining three iGEM restriction sites EcoRI (1578),
 +
PstI
 +
(1773) and EcoRI (1796) were replaced by a synthetic gene fragment,
 +
since the <i>rep
 +
</i>ORF contained these restriction sites in close proximity to
 +
each other plus
 +
an additional KpnI restriction site which was also not desired (see </span><span
 +
lang="EN-US">Figure 2</span><span lang="EN-US">).
 +
This gene fragment was cloned into
 +
the <i>rep</i> gene using HindIII and SwaI, which are
 +
single-cutting
 +
restriction enzymes adjacent to the target area. Additionally, BamHI
 +
(859) and
 +
SalI (1239) were removed, because these enzymes were required for
 +
genetically
 +
inserting the loop modifications in VP123.</span></p>
 +
<h4 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275885925"></a><a
 +
name="_Toc275817884"><span lang="EN-US">Modifications in VP123</span></a></h4>
 +
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">In
 +
order to
 +
implement the restriction sites necessary for targeting via loop
 +
insertions,
 +
the gene coding for the VP proteins was modified as well. The
 +
introduction of
 +
these restriction required up to four base mutations in a row, hence it
 +
was
 +
decided to synthesize this gene fragment and replace the wild-type
 +
sequence in <i>RepVP123</i>
 +
as well.</span></p>
 +
<table class="MsoTableGrid"
 +
style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;"
 +
border="0" cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr>
 +
<td
 +
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 460.6pt;">
 +
<p class="MsoNormal"
 +
style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
 +
align="center"><img style="width: 574px; height: 218px;" id="Picture 5"
 +
src="https://static.igem.org/mediawiki/2010/c/cd/Freiburg10_cap_synthetic_gene_fragment.png"
 +
alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\Cap_synthesis_marked.jpg"></p>
 +
<p class="MsoCaption"><span lang="EN-US">Figure </span><span
 +
lang="EN-US">4</span> <span
 +
style="font-size: 8pt; color: windowtext; font-weight: normal;"
 +
lang="EN-US">Restriction sites within <i>cap</i>
 +
sequence showing introduced loop insertion restriction sites into <i>cap</i>
 +
to enable cloning of targeting or purification motifs into both 453 and
 +
587 loops. Again, the red box indicates gene sequence which was
 +
synthetized.</span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
 +
<table class="MsoTableGrid"
 +
style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;"
 +
border="0" cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr>
 +
<td
 +
style="border: 1pt solid windowtext; padding: 0cm 3.5pt; vertical-align: top; width: 460.6pt;">
 +
<p class="MsoNormal"
 +
style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
 +
align="center"><img style="width: 511px; height: 302px;" alt=""
 +
id="Chart 7"
 +
src="https://static.igem.org/mediawiki/2010/6/6b/Freiburg10_wt_vs_4xmutant.png"></p>
 +
<p class="MsoCaption"><span lang="EN-US">Figure </span><span
 +
lang="EN-US">5</span> <span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">Results
 +
for transduction efficiency measured by flow cytometry. Fluorescence is
 +
measured in surviving cells. The tested <i>RepVP123</i>
 +
containing four point mutation to delete iGEM and loop insertion
 +
restriction sites does not show any difference within mVenus expression
 +
compared to the wild-type and therefore can be verified working.</span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">Alongside
 +
to
 +
creating an iGEM compatible plasmid, infectivity of the modified
 +
construct was
 +
tested in cell culture via flow cytometry. Then experiments confirmed
 +
that
 +
single cloning steps did not interfere with natural viral infectivity
 +
at first
 +
(see above). But cloning of the synthesized <i>rep</i>
 +
gene fragment into the
 +
plasmid dramatically reduced transduction efficiency as detected by
 +
flow
 +
cytometry. Scrutinizing each mutation and its potential impact,
 +
suggested that
 +
abolished transduction was related to the mutation, which removed the
 +
KpnI
 +
(1721) site. This site is located within a splice site, which is
 +
crucial for
 +
the Rep proteins, and thus even silent mutations may interfere with
 +
virus
 +
production (see extra topic “Rep proteins”).</span></p>
 +
<span
 +
style="font-size: 11pt; line-height: 200%; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
 +
lang="EN-US"><br style="page-break-before: always;" clear="all">
 +
</span>
 +
<p class="MsoNormal" style="text-indent: 0cm; text-align: center;"><span
 +
lang="EN-US">&nbsp;<img alt="xxx"
 +
src="https://static.igem.org/mediawiki/2010/f/fd/Freiburg10_Freiburg10_facs_table1.png"><br>
 +
</span></p>
 +
<br>
 +
<p style="text-align: center;" class="MsoNormal">&nbsp;<img
 +
style="width: 636px; height: 396px;" alt=""
 +
src="https://static.igem.org/mediawiki/2010/3/3a/Freiburg10_Freiburg10_facs_table2.png"></p>
 +
<br>
 +
<p class="MsoNormal" style="text-indent: 0cm; line-height: 150%;"><span
 +
lang="EN-US"></span></p>
 +
<p class="MsoNormal" style="text-indent: 0cm; line-height: 150%;"><span
 +
lang="EN-US"></span></p>
 +
<table
 +
style="width: 474px; text-align: left; margin-left: auto; margin-right: auto;"
 +
border="0" cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr>
 +
<td style="width: 464px;">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 10pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
 +
align="center"><span style="font-size: 10pt;"><img
 +
style="width: 395px; height: 215px;" id="Chart 18"
 +
src="https://static.igem.org/mediawiki/2010/7/73/Freiburg10_KpnI_deleted.png"
 +
alt="Description: Fluorescence of HT1080 cells transduced with FRAGE AN ADRIAN:WELCHES GOI? and P325 and P326 both containing RepCap in which the synthetized gene sequences were inserted. As control,pAAV_RC (P50) containing wtRepCap was used. mVenus expression within cells clearly state that tested constructs does not meet expectations."></span></p>
 +
<p style="text-align: justify;" class="MsoCaption"><span
 +
lang="EN-US">Figure </span><span lang="EN-US">8</span> <span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">AAV-293
 +
cells were transfected with three plasmids pHelper,
 +
pSB1C3_[AAV2]-left-ITR_pCMV_betaglobin_mVenus _hGH_[AAV2]-right-ITR and
 +
pAAV_RC_inserts (see above) or pAAV_RC (see below) providing essential
 +
genes and proteins for producing viral particles. After 48 hour post
 +
transfection, viral particles were harvested by freeze-thaw lysis and
 +
centrifugation followed by HT1080 transduction. mVenus expression of
 +
viral genomes was determined by flow cytomery 24 hours post
 +
infection. </span><span style="color: windowtext; font-weight: normal;"
 +
lang="EN-US">Fluorescence
 +
is measured in surviving cells</span><span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">.
 +
Results show that insertion of both <i>rep</i>
 +
and <i>cap</i> syntheses disrupts viral infectivity.</span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<p class="MsoNormal" style="text-indent: 0cm; line-height: 150%;"><span
 +
lang="EN-US"></span></p>
 +
<p class="MsoNormal" style="text-indent: 0cm; line-height: 150%;"><span
 +
lang="EN-US">Therefore,
 +
additional constructs were designed containing only the synthetic <i>cap</i>
 +
sequence or both, the synthesized sequences plus a re-mutation of KpnI
 +
(1721), in
 +
order to re-establish the wild-type splice site within the <i>rep</i>
 +
ORF. Results
 +
from cell culture obtained via FACS revealed that in fact the poor
 +
results were
 +
related to the KpnI restriction site deletion. Both constructs showed a
 +
transduction efficiency corresponding to the unmodified wild-type <i>RepVP123</i>’s
 +
transduction efficiency.</span></p>
 +
<div align="center">
 +
<table class="MsoTableGrid"
 +
style="border: medium none ; border-collapse: collapse;" border="0"
 +
cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr style="height: 177.6pt;">
 +
<td
 +
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 464.4pt; height: 177.6pt;">
 +
<p class="MsoNormal"
 +
style="margin-bottom: 10pt; text-align: center; text-indent: 0cm; line-height: 115%; page-break-after: avoid;"
 +
align="center"><img style="width: 625px; height: 233px;" alt=""
 +
id="Chart 3"
 +
src="https://static.igem.org/mediawiki/2010/6/62/Freiburg10_KpnI_reinstated.png"></p>
 +
<p class="MsoCaption"><span lang="EN-US">Figure </span><span
 +
lang="EN-US">9</span> <span lang="EN-US">F</span><span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">luorescence
 +
of cells transduced with mVenus carrying rAAV measured by flow
 +
cytometry. AAV-293 cells were transfected with three plasmids pHelper,
 +
pSB1C3_[AAV2]-left-ITR_pCMV_betaglobin_mVenus_hGH_[AAV2]-right-ITR and <i>RepVP123</i>
 +
constructs providing essential genes and proteins for producing viral
 +
particles. 48 hours post transfection, viral particles were
 +
harvested by freeze-thaw lysis and centrifugation followed by HT1080
 +
transduction. mVenus expression of viral genomes was determined by flow
 +
cytometry 24 hours post infection. Results show that <i>cap </i>integration
 +
does not influence infectivity. </span><span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">Fluorescence
 +
is measured in surviving cells.</span><span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">
 +
Recreation
 +
of KpnI within <i>rep</i> splice site recovers
 +
transduction efficiency.</span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
<p class="MsoNormal" style="text-indent: 0cm; line-height: 150%;"><span
 +
lang="EN-US">&nbsp;</span></p>
 +
<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275885926"></a><a
 +
name="_Toc275817885"><span lang="EN-US">Modularization: Adapting
 +
pSB1C3 to loop
 +
insertions – pSB1C3_001</span></a></h3>
 +
<p class="MsoNormal" style="text-indent: 0cm; line-height: 150%;"><span
 +
lang="EN-US">To
 +
fulfill iGEM requirements all plasmids need to be submitted in pSB1C3,
 +
therefore primers were ordered for amplifying <i>RepVP123</i>
 +
containing all
 +
modifications done so far by PCR and cloning the into pSB1C3. Still,
 +
pSB1C3
 +
contains two restriction sites for SspI and PvuII restriction enzymes
 +
in its
 +
CAT marker. Since these are necessary for cloning ViralBricks in this
 +
vector,
 +
the iGEM Team Freiburg_Bioware 2010 decided in agreement with iGEM
 +
Headquarters
 +
to implement a new standard for the pSB1C3 backbone which was named
 +
pSB1C3_001.
 +
Both restriction sites interfering with ViralBrick insertions were
 +
mutated to
 +
make SspI and PvuII single-cutters (see method development).</span></p>
 +
<table class="MsoTableGrid"
 +
style="border: medium none ; margin-left: auto; border-collapse: collapse; text-align: left; margin-right: auto;"
 +
border="0" cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr>
 +
<td
 +
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 460.6pt;">
 +
<p class="MsoNormal"
 +
style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
 +
align="center"><img style="width: 584px; height: 278px;"
 +
id="Picture 16"
 +
src="https://static.igem.org/mediawiki/2010/d/d1/Freiburg10_pSB1C3_001_mutations.png"
 +
alt=""></p>
 +
<p class="MsoCaption"><span lang="EN-US">Figure </span><span
 +
lang="EN-US">10</span> <span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">Comparison
 +
of pSB1C3 (upper row) and pSB1C3_001 (lower row). Deletions of SspI and
 +
PvuII are marked by red boxes.</span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<p class="MsoNormal"><i><span lang="EN-US">RepVP123</span></i><span
 +
lang="EN-US">
 +
containing both <i>rep</i> and <i>cap</i>
 +
synthetic gene fragments including
 +
the re-mutation of KpnI and the downstream p5TATA-less promotor was
 +
cloned into
 +
the newly constructed pSB1C3_001. Testing this newly assembled plasmid
 +
in cell
 +
culture revealed unexpected data. Not only did the newly assembled
 +
plasmid work
 +
(see Figure 10), but in comparison to pAAV containing the same <i>RepVP123</i>
 +
construct, pSB1C3_001 showed an about 3 times higher transduction
 +
efficiency.
 +
Although exact reasons are still unknown, these results are probably
 +
related to
 +
the reduced length of pSB1C3_001 compared to the original pAAV plasmid
 +
of
 +
approximately 1000 base pairs.</span></p>
 +
<span
 +
style="font-size: 11pt; line-height: 200%; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
 +
lang="EN-US"></span><span lang="EN-US">&nbsp;</span>
 +
<table
 +
style="border: medium none ; width: 466px; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;"
 +
class="MsoTableGrid" border="0" cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr style="height: 209.85pt;">
 +
<td
 +
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 4661px; height: 209.85pt;">
 +
<p class="MsoNormal"
 +
style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
 +
align="center"><span style="font-size: 10pt; line-height: 200%;"><img
 +
style="width: 516px; height: 258px;" alt="" id="Chart 15"
 +
src="https://static.igem.org/mediawiki/2010/2/2b/Freiburg10_pAAV_pSB1C3_001.png"></span></p>
 +
<p class="MsoCaption"><span lang="EN-US">Figure </span><span
 +
lang="EN-US">11</span> <span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">AAV-293
 +
cells were transfected with three plasmids pHelper,
 +
pSB1C3_001_[AAV2]-Rep-VP123_p5-TATAless or pAAV_RC_IRCK and
 +
pSB1C3_[AAV2]-left-ITR_pCMV_beta-globin_mVenus_hGH_[AAV2]-right-ITR
 +
providing essential genes and proteins for producing viral particles.
 +
After 48 hours post transfection, viral particles were harvested by
 +
freeze-thaw lysis and centrifugation followed by HT1080 transduction.
 +
mVenus expression of viral genomes was determined by flow cytomery
 +
after 24 hours post infection. </span><span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">Fluorescence
 +
is measured in surviving cells.</span><span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">&nbsp;Results
 +
showed functionality of <i>RepVP123</i>
 +
within pSB1C3_001 vector and additionally increased transduction
 +
efficiency.</span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<h4 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275885927"></a><a
 +
name="_Toc275817886"><span lang="EN-US">Turning-off natural tropism:
 +
HSPG-knock-out</span></a></h4>
 +
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">Shutting-down
 +
the
 +
natural viral tropism is essential for targeting specifically tumor
 +
cells and
 +
not infecting healthy cells. Therefore, the iGEM team Freiburg_Bioware
 +
2010
 +
decided to knock-out the viral natural tropism delivered by the heperan
 +
sulfate
 +
proteoglycan-(HSPG) binding site within the viruses 587 loop. The
 +
knock-out was
 +
cloned by designing primers containing the required base exchanges and
 +
performing a SDM. Like performed before, this <i>RepVP123</i>
 +
variant was tested
 +
in cell culture as well and evaluated by flow cytometry. Results show
 +
that
 +
mutation of HSPG-binding motif has severe impact on transduction
 +
efficiency
 +
thus enabling a viral particle carrying this knock-out and additional
 +
targeting
 +
motifs, e.g. within the loops or presented via N-terminal fusion to
 +
bind target
 +
cells’ receptors and therefore infecting target cells at a much higher
 +
rate
 +
compared to unspecific infection of other cell types within an organism
 +
(see </span><span lang="EN-US">Figure 12</span><span lang="EN-US">).</span></p>
 +
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">To
 +
quantify
 +
differences in infectivity, the infectious titer of viral particles
 +
built-up of
 +
<i>RepVP123</i> with and without HSPG binding motif was
 +
determined by qPCR (see
 +
</span><span lang="EN-US">Figure 14</span><span lang="EN-US">) for
 +
different cell
 +
lines. Results show that the implemented HSPG-knock-out verifies
 +
results obtained
 +
from flow cytometry, infectious titers severely compared to <i>RepVP123</i>
 +
with intact HSPG binding motif.</span></p>
 +
<table class="MsoTableGrid"
 +
style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;"
 +
border="0" cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr style="height: 258.85pt;">
 +
<td
 +
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 459.85pt; height: 258.85pt;">
 +
<p class="MsoNormal"
 +
style="text-indent: 0cm; page-break-after: avoid;"><img
 +
style="width: 595px; height: 379px;" id="Picture 2"
 +
src="https://static.igem.org/mediawiki/2010/9/9d/Freiburg10_HSPG-ko_sequence.png"
 +
alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\HSPG-ko_modified.jpg"></p>
 +
<p class="MsoCaption" style="text-align: left;" align="left"><span
 +
lang="EN-US">Figure </span><span lang="EN-US">12</span> <span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">Alignment
 +
of 587 loop within viral VP123: The upper sequence shows a strand
 +
containing the HSPG binding motif (AGA, in red boxes), the lower
 +
sequence contains the HSPG-ko introduced by the iGEM team
 +
Freiburg_Bioware 2010 (GCT and GCC, blue boxes).</span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
 +
<table class="MsoTableGrid"
 +
style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;"
 +
border="0" cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr>
 +
<td
 +
style="border: 1pt solid windowtext; padding: 0cm 3.5pt; vertical-align: top; width: 460.6pt;">
 +
<p class="MsoNormal"
 +
style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
 +
align="center"><img style="width: 467px; height: 229px;" alt=""
 +
id="Chart 9"
 +
src="https://static.igem.org/mediawiki/2010/0/01/Freiburg10_columns_HSPG_HSPG_ko.png"></p>
 +
<p class="MsoCaption"><a name="_Ref275882130"><span lang="EN-US">Figure
 +
</span></a><span lang="EN-US">13</span> <span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">Transduction
 +
efficiency of HT1080 cells measured by flow cytometry.&nbsp;</span><span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">Fluorescence
 +
is measured in surviving cells.</span><span
 +
style="color: windowtext; font-weight: normal;" lang="EN-US">
 +
Knock-out of
 +
HSPG binding motif greatly reduces transduction efficiency compared to <i>RepVP123</i>
 +
containing the motiv.</span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
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Latest revision as of 02:45, 28 October 2010

Modularization


Contents

Modularization of the vectorplasmid

Introduction to Modularization of Vectorplasmid. 1

Recombinant and Modular Vectorplasmid Carrying GOI 2

Cloning and Combination Strategies for the Vectorplasmid. 3

Testing functionality of Assembled Vectorplasmid. 7

Fluorescence Microscopy of Target Cells Demonstrates GOI Expression. 7

Analysis of Target Cells by Flow Cytometry demonstrates GOI Expression. 8

Influence of hGH terminator BioBrick on GOI Expression. 8

Influence of Beta-globin intron Biobrick on GOI Expression. 11

Functionality of the Full Assembled Vectorplasmid Demonstrated by GOI Expression. 14

Conclusion. 16

References. 17

 

Modularization of the RepVP123 plasmids

Overview of RepVP123 plasmid. 2

Modularization: Overview.. 2

Modularization: Removing iGEM restriction sites and establishing loop insertion capability. 3

Modifications in Rep. 3

Modifications in VP123. 4

Modularization: Adapting pSB1C3 to loop insertions – pSB1C3_001. 7

Turning-off natural tropism: HSPG-knock-out. 9

 


Introduction to Modularization of vector plasmid


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 Vectorplasmid 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_K40410), 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).

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 ‘Method Development of Cloning Strategy for ITRs’).

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.

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

Figure 3: 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.

Figure 4: 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.

 

Beschreibung: \\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 5: 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).

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

Figure 6: 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.

A

B

C

D

Figure 7: 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

Vectorplasmid containing hGH terminator signal

Figure 8: 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.

 

 

Figure 9: Flow cytometry analysis of vectorplasmids with and without hGH terminator. YFP expression of viral genomes was determined by flow cytomery 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

Vectorplasmid containing beta-globin intron

Figure 10: 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.

 

Figure 11: 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
Producing recombinant virus particles for therapeutical applications is, besides specific cell targeting, 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 biological system of the Adeno-associated virus serotype 2 was examined by an exhaustive literature search. Subsequently, the essential components for AAV-2 particle production were extracted and redesigned to match the iGEM standard.
The provided tripartite system is independent of a superinfection  of Adeno- or herpes simplex viruses since the genes encoding the required helper-proteins are co-transfected. Inside the eukaryotic host cell, the DNA sequence containing the inverted terminal repeats (ITRs) is extracted and later encapsidated into the preformed capsids after production of single-stranded DNA. Consequently, this plasmid is known as the vector plasmid (pGOI). Promoter, beta-globin intron and the hGH terminator signal are flanked by the ITRs (ITRs, BBa_K404100 and BBa_K404101) and regulate transgene expression. The vector plasmid containing the desired gene of interest is cotransfected with the RepCap plasmid ( BBa_K404001, BBa_K404102 or BBa_K404103) and the pHelper plasmid. To obtain the fully assembled vector plasmid, several assembly steps have to be performed.  
After assembly of plasmids containing all required elements, vector plasmid functionality was confirmed in cell culture. AAV-293 cells stably expressing the 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).
In the beginning, the iGEM team Freiburg_Bioware 2010 used a commercial vector plasmid, pAAV-MCS (Stratagene), to determine whether virus particle production by AAV-293 cells could be achieved. iGEM RFC 25 restriction enzyme sites were introduced and the fluorescent protein mVenus was subcloned into this plasmid. Subsequently, this plasmid was used as a reference and compared to the assembled vector plasmid pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (abbreviated 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 reference plasmid and the pSB1C3_mVenus plasmid revealed that biological functionality of the reassembled plasmid was preserved.

pSB1C3_mVenus ( BBa_K404119)

pAAV_mVenus (reference)

Figure 12: Flow cytometry analysis of fluorescent protein expression in transduced HT1080 cells. For viral particle production, AAV-293 cells were transfected with the reassembled vector plasmid ( BBa_K404119) or the reference plasmid, respectively. A: Gating non transduced cells (control); subcellular debris and cellular aggreates 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 cells expressing 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) (plasmids used for transfection: pGOI: pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (pSB1C3_mVenus: BBa_K404119), pHelper, pRC. D: Transduced cells plotted against cells expressing mVenus. R10 comprises transduced cells detected by mVenus fluorescence. E: Overlay of non-transduced (red) and transduced (green). F: Gating non-transduced cells (control). G: Non-transduced cells plotted against cells expressing mVenus (R5). H: Gating transduced cells (R14 ≙ R2) (plasmids used for transfection: pGOI: pAAV_mVenus, pHelper). I: Transduced cells plotted against cells expressing mVenus. R10 comprises transduced cells detected by mVenus fluorescence. J: Overlay of non-transduced (red) and transduced (green) cells plotted against mVenus expression. 

 

 

 

Figure 13: Quantification of mVenus fluorescence by flow cytometry analysis. The data source is identical to Figure 13. Compared to the reference vector plasmid, mVenus expression in HT1080 cells is enhanced in case of the assembled plasmid (pSB1C3_mVenus) 

Conclusion

Idea of the modular ‘Virus Construction Kit’ is to provide all required elements for producing recombinant, functional virus particles delivering encapsidated genes of interest to specific cells. First step was to modify and modularize the vectorplasmid comprising basically the cis-elements for replication (ITRs), a strong eukaryotic or tissue specific promoter (pCMV or phTERT), the gene of interest (fluorescent proteins or suicide genes) and the hGH termination signal. Each element was successfully cloned and reassembled resulting in functional vectorplasmids determined by flow cytometry and fluorescence microscopy analyses. Experiments have been performed with mVenus since measurement of fluorescent proteins can be easily performed and visualized. Considering the results, the iGEM team Freiburg_Bioware 2010 then tested the construct containing the suicide genes thymidine kinase and cytosine deaminase. Further details demonstrating efficient tumor killing, using prodrug-activating systems, see results page Arming.

References

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), 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), 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), 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), 607-17. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12702819.

 


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Overview of RepVP123 plasmid

Modularization: Overview

In our terminology the term “RepVP123” encompasses the whole AAV2 genome excluding the ITRs. The rep locus comprises four proteins related to genome replication while the cap locus codes for the proteins VP1, VP2, VP3 and the assembly-associated protein (AAP), which are required for viral capsid assembly. Source of the RepVP123 BioBrick supplied within iGEM team Freiburg_Bioware 2010 Virus Construction Kit is the wild-type AAV2 RepVP123, as provided e. g. in the pAAV vector from Stratagene. In order to introduce iGEM standard and additionally enabling the possibility to modify the viral capsid via integration of certain motives within the viral loops 453 and 587 a total of twelve mutations within RepVP123 (see Figure 1) and additionally two mutations within the pSB1C3 backbone were performed either by Site-Directed Mutagenesis (SDM) or by ordering and cloning of specifically designed gene sequences matching the required demands. Modifying the pSB1C3 led to iGEM team Freiburg_Bioware’s variant of this backbone, pSB1C3_001.

Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\RepCap_complete_modifications_arrows.jpg

Figure 1 Mutations implemented into RepVP123 in order to establish both iGEM standard and loop insertion capability. Green arrows indicate integrated restriction sites, red red arrows indicate deleted restriction sites. KpnI was deleted first and reinstated afterwards. (see text).


 

Plasmid name:

Functionality (determinded in cell culture via transduction and flow cytometry ):

4x mutations (PstI (310), BamHI (859), SalI (1239), PstI (4073))

inserted rep fragment

inserted cap fragment

reinstated KpnI

pAAV

pSB1C3_001

HSPG-ko

pAAV_RC (wild-type)

yes

 

 

 

 

x

 

 

pAAV_RC_4x mutant

yes

x

 

 

 

x

 

 

pAAV_RC_inserts

no

x

x

x

 

x

 

 

pAAV_RC_Cap

yes

x

 

x

 

x

 

 

pAAV_RC_RepVP123

yes

x

x

x

x

x

 

 

pSB1C3_RepVP123_ p5TATAless

yes

x

x

x

x

 

x

 

pSB1C3_RepVP123_ HSPG-ko_p5TATAless

yes

x

x

x

x

 

x

x

Figure 2 Table contains complete overview about all plasmids containing RepVP123 which were used by iGEM team Freiburg_Bioware 2010.

Modularization: Removing iGEM restriction sites and establishing loop insertion capability

Modifications in Rep

Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\Rep_synthesis_marked.jpg

Figure 3 Restriction sites within the wild-type rep gene sequence, which were removed via cloning of synthetized rep gene fragment into the plasmid. The red box indicates the region spanned by the synthetic sequence.

Making the RepVP123 wild-type compatible with the iGEM standards required the removal of five restriction sites (see Figure 1). This was achieved using site-directed mutagenesis for PstI (position 310) and PstI (4073). The remaining three iGEM restriction sites EcoRI (1578), PstI (1773) and EcoRI (1796) were replaced by a synthetic gene fragment, since the rep ORF contained these restriction sites in close proximity to each other plus an additional KpnI restriction site which was also not desired (see Figure 2). This gene fragment was cloned into the rep gene using HindIII and SwaI, which are single-cutting restriction enzymes adjacent to the target area. Additionally, BamHI (859) and SalI (1239) were removed, because these enzymes were required for genetically inserting the loop modifications in VP123.

Modifications in VP123

In order to implement the restriction sites necessary for targeting via loop insertions, the gene coding for the VP proteins was modified as well. The introduction of these restriction required up to four base mutations in a row, hence it was decided to synthesize this gene fragment and replace the wild-type sequence in RepVP123 as well.

Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\Cap_synthesis_marked.jpg

Figure 4 Restriction sites within cap sequence showing introduced loop insertion restriction sites into cap to enable cloning of targeting or purification motifs into both 453 and 587 loops. Again, the red box indicates gene sequence which was synthetized.

 

Figure 5 Results for transduction efficiency measured by flow cytometry. Fluorescence is measured in surviving cells. The tested RepVP123 containing four point mutation to delete iGEM and loop insertion restriction sites does not show any difference within mVenus expression compared to the wild-type and therefore can be verified working.

Alongside to creating an iGEM compatible plasmid, infectivity of the modified construct was tested in cell culture via flow cytometry. Then experiments confirmed that single cloning steps did not interfere with natural viral infectivity at first (see above). But cloning of the synthesized rep gene fragment into the plasmid dramatically reduced transduction efficiency as detected by flow cytometry. Scrutinizing each mutation and its potential impact, suggested that abolished transduction was related to the mutation, which removed the KpnI (1721) site. This site is located within a splice site, which is crucial for the Rep proteins, and thus even silent mutations may interfere with virus production (see extra topic “Rep proteins”).


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Description: Fluorescence of HT1080 cells transduced with FRAGE AN ADRIAN:WELCHES GOI? and P325 and P326 both containing RepCap in which the synthetized gene sequences were inserted. As control,pAAV_RC (P50) containing wtRepCap was used. mVenus expression within cells clearly state that tested constructs does not meet expectations.

Figure 8 AAV-293 cells were transfected with three plasmids pHelper, pSB1C3_[AAV2]-left-ITR_pCMV_betaglobin_mVenus _hGH_[AAV2]-right-ITR and pAAV_RC_inserts (see above) or pAAV_RC (see below) providing essential genes and proteins for producing viral particles. After 48 hour post transfection, viral particles were harvested by freeze-thaw lysis and centrifugation followed by HT1080 transduction. mVenus expression of viral genomes was determined by flow cytomery 24 hours post infection. Fluorescence is measured in surviving cells. Results show that insertion of both rep and cap syntheses disrupts viral infectivity.

Therefore, additional constructs were designed containing only the synthetic cap sequence or both, the synthesized sequences plus a re-mutation of KpnI (1721), in order to re-establish the wild-type splice site within the rep ORF. Results from cell culture obtained via FACS revealed that in fact the poor results were related to the KpnI restriction site deletion. Both constructs showed a transduction efficiency corresponding to the unmodified wild-type RepVP123’s transduction efficiency.

Figure 9 Fluorescence of cells transduced with mVenus carrying rAAV measured by flow cytometry. AAV-293 cells were transfected with three plasmids pHelper, pSB1C3_[AAV2]-left-ITR_pCMV_betaglobin_mVenus_hGH_[AAV2]-right-ITR and RepVP123 constructs providing essential genes and proteins for producing viral particles. 48 hours post transfection, viral particles were harvested by freeze-thaw lysis and centrifugation followed by HT1080 transduction. mVenus expression of viral genomes was determined by flow cytometry 24 hours post infection. Results show that cap integration does not influence infectivity. Fluorescence is measured in surviving cells. Recreation of KpnI within rep splice site recovers transduction efficiency.

 

Modularization: Adapting pSB1C3 to loop insertions – pSB1C3_001

To fulfill iGEM requirements all plasmids need to be submitted in pSB1C3, therefore primers were ordered for amplifying RepVP123 containing all modifications done so far by PCR and cloning the into pSB1C3. Still, pSB1C3 contains two restriction sites for SspI and PvuII restriction enzymes in its CAT marker. Since these are necessary for cloning ViralBricks in this vector, the iGEM Team Freiburg_Bioware 2010 decided in agreement with iGEM Headquarters to implement a new standard for the pSB1C3 backbone which was named pSB1C3_001. Both restriction sites interfering with ViralBrick insertions were mutated to make SspI and PvuII single-cutters (see method development).

Figure 10 Comparison of pSB1C3 (upper row) and pSB1C3_001 (lower row). Deletions of SspI and PvuII are marked by red boxes.

RepVP123 containing both rep and cap synthetic gene fragments including the re-mutation of KpnI and the downstream p5TATA-less promotor was cloned into the newly constructed pSB1C3_001. Testing this newly assembled plasmid in cell culture revealed unexpected data. Not only did the newly assembled plasmid work (see Figure 10), but in comparison to pAAV containing the same RepVP123 construct, pSB1C3_001 showed an about 3 times higher transduction efficiency. Although exact reasons are still unknown, these results are probably related to the reduced length of pSB1C3_001 compared to the original pAAV plasmid of approximately 1000 base pairs.

 

Figure 11 AAV-293 cells were transfected with three plasmids pHelper, pSB1C3_001_[AAV2]-Rep-VP123_p5-TATAless or pAAV_RC_IRCK and pSB1C3_[AAV2]-left-ITR_pCMV_beta-globin_mVenus_hGH_[AAV2]-right-ITR providing essential genes and proteins for producing viral particles. After 48 hours post transfection, viral particles were harvested by freeze-thaw lysis and centrifugation followed by HT1080 transduction. mVenus expression of viral genomes was determined by flow cytomery after 24 hours post infection. Fluorescence is measured in surviving cells. Results showed functionality of RepVP123 within pSB1C3_001 vector and additionally increased transduction efficiency.

Turning-off natural tropism: HSPG-knock-out

Shutting-down the natural viral tropism is essential for targeting specifically tumor cells and not infecting healthy cells. Therefore, the iGEM team Freiburg_Bioware 2010 decided to knock-out the viral natural tropism delivered by the heperan sulfate proteoglycan-(HSPG) binding site within the viruses 587 loop. The knock-out was cloned by designing primers containing the required base exchanges and performing a SDM. Like performed before, this RepVP123 variant was tested in cell culture as well and evaluated by flow cytometry. Results show that mutation of HSPG-binding motif has severe impact on transduction efficiency thus enabling a viral particle carrying this knock-out and additional targeting motifs, e.g. within the loops or presented via N-terminal fusion to bind target cells’ receptors and therefore infecting target cells at a much higher rate compared to unspecific infection of other cell types within an organism (see Figure 12).

To quantify differences in infectivity, the infectious titer of viral particles built-up of RepVP123 with and without HSPG binding motif was determined by qPCR (see Figure 14) for different cell lines. Results show that the implemented HSPG-knock-out verifies results obtained from flow cytometry, infectious titers severely compared to RepVP123 with intact HSPG binding motif.

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Figure 12 Alignment of 587 loop within viral VP123: The upper sequence shows a strand containing the HSPG binding motif (AGA, in red boxes), the lower sequence contains the HSPG-ko introduced by the iGEM team Freiburg_Bioware 2010 (GCT and GCC, blue boxes).

 

Figure 13 Transduction efficiency of HT1080 cells measured by flow cytometry. Fluorescence is measured in surviving cells. Knock-out of HSPG binding motif greatly reduces transduction efficiency compared to RepVP123 containing the motiv.

 

Figure 14 Infectious titers of RepVP123 with and without natural HSPG binding motif tested in different cell lines via qPCR. Shutting-down the HSPG binding motif reduces infectious titer in both HT1080 and HeLa cell lines. For A431 cells, no infectious titer could be detected via qPCR, which is probably related to poor transduction efficiency of A431 cells.

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