Team:Freiburg Bioware/Project/Results/Modularization Vector Plasmid
From 2010.igem.org
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<html> | <html> | ||
+ | <head> | ||
+ | </head> | ||
+ | <body> | ||
<h1>Modularization</h1> | <h1>Modularization</h1> | ||
<br> | <br> | ||
<h2>Contents</h2> | <h2>Contents</h2> | ||
<h3>Modularization of the vectorplasmid</h3> | <h3>Modularization of the vectorplasmid</h3> | ||
- | <p class="MsoToc2"><a href="#_Toc275800680"><span lang="EN-US">Introduction | + | <p class="MsoToc2"><a href="#_Toc275800680"><span |
+ | lang="EN-US">Introduction | ||
to | to | ||
Modularization of Vectorplasmid</span><span | Modularization of Vectorplasmid</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;">1</span></a></p> | + | </span><span |
- | <p class="MsoToc3"><a href="#_Toc275800681"><span lang="EN-US">Recombinant | + | style="color: windowtext; display: none; text-decoration: none;">1</span></a></p> |
+ | <p class="MsoToc3"><a href="#_Toc275800681"><span | ||
+ | lang="EN-US">Recombinant | ||
and | and | ||
Modular Vectorplasmid Carrying GOI</span><span | Modular Vectorplasmid Carrying GOI</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;">2</span></a></p> | + | </span><span |
- | <p class="MsoToc3"><a href="#_Toc275800682"><span lang="EN-US">Cloning | + | style="color: windowtext; display: none; text-decoration: none;">2</span></a></p> |
+ | <p class="MsoToc3"><a href="#_Toc275800682"><span | ||
+ | lang="EN-US">Cloning | ||
and | and | ||
Combination Strategies for the Vectorplasmid</span><span | Combination Strategies for the Vectorplasmid</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;">3</span></a></p> | + | </span><span |
- | <p class="MsoToc3"><a href="#_Toc275800683"><span lang="EN-US">Testing | + | style="color: windowtext; display: none; text-decoration: none;">3</span></a></p> |
+ | <p class="MsoToc3"><a href="#_Toc275800683"><span | ||
+ | lang="EN-US">Testing | ||
functionality of Assembled Vectorplasmid</span><span | functionality of Assembled Vectorplasmid</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;">7</span></a></p> | + | </span><span |
- | <p class="MsoToc4"><a href="#_Toc275800684"><span lang="EN-US">Fluorescence | + | style="color: windowtext; display: none; text-decoration: none;">7</span></a></p> |
+ | <p class="MsoToc4"><a href="#_Toc275800684"><span | ||
+ | lang="EN-US">Fluorescence | ||
Microscopy of Target Cells Demonstrates GOI Expression</span><span | Microscopy of Target Cells Demonstrates 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;">7</span></a></p> | + | </span><span |
- | <p class="MsoToc4"><a href="#_Toc275800685"><span lang="EN-US">Analysis | + | style="color: windowtext; display: none; text-decoration: none;">7</span></a></p> |
+ | <p class="MsoToc4"><a href="#_Toc275800685"><span | ||
+ | lang="EN-US">Analysis | ||
of Target | of Target | ||
Cells by Flow Cytometry demonstrates GOI Expression</span><span | Cells by Flow Cytometry demonstrates 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;">8</span></a></p> | + | </span><span |
- | <p class="MsoToc5"><a href="#_Toc275800686"><span lang="EN-US">Influence | + | style="color: windowtext; display: none; text-decoration: none;">8</span></a></p> |
+ | <p class="MsoToc5"><a href="#_Toc275800686"><span | ||
+ | lang="EN-US">Influence | ||
of hGH | of hGH | ||
terminator BioBrick on GOI Expression</span><span | terminator 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;">8</span></a></p> | + | </span><span |
- | <p class="MsoToc5"><a href="#_Toc275800687"><span lang="EN-US">Influence | + | style="color: windowtext; display: none; text-decoration: none;">8</span></a></p> |
+ | <p class="MsoToc5"><a href="#_Toc275800687"><span | ||
+ | lang="EN-US">Influence | ||
of <i>Beta-globin</i> | of <i>Beta-globin</i> | ||
intron Biobrick on GOI Expression</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;">11</span></a></p> | + | </span><span |
- | <p class="MsoToc5"><a href="#_Toc275800688"><span lang="EN-US">Functionality | + | style="color: windowtext; display: none; text-decoration: none;">11</span></a></p> |
+ | <p class="MsoToc5"><a href="#_Toc275800688"><span | ||
+ | lang="EN-US">Functionality | ||
of the | of the | ||
Full Assembled Vectorplasmid Demonstrated by GOI Expression</span><span | 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;">. |
- | style="color: windowtext; display: none; text-decoration: none;">14</span></a></p> | + | </span><span |
- | <p class="MsoToc3"><a href="#_Toc275800689"><span lang="EN-US">Conclusion</span><span | + | style="color: windowtext; display: none; text-decoration: none;">14</span></a></p> |
- | style="color: windowtext; display: none; text-decoration: none;">. </span><span | + | <p class="MsoToc3"><a href="#_Toc275800689"><span |
- | style="color: windowtext; display: none; text-decoration: none;">16</span></a></p> | + | lang="EN-US">Conclusion</span><span |
- | <p class="MsoToc3"><a href="#_Toc275800690"><span lang="EN-US">References</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;">17</span></a></p> | + | 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> </b></a></p> | <p class="MsoNormal"><a name="_Toc275797953"><b> </b></a></p> | ||
<h3>Modularization of the RepVP123 plasmids</h3> | <h3>Modularization of the RepVP123 plasmids</h3> | ||
<div class="WordSection1"> | <div class="WordSection1"> | ||
- | <p class="MsoToc2"><a href="#_Toc275885921"><span lang="EN-US">Overview | + | <p class="MsoToc2"><a href="#_Toc275885921"><span |
+ | lang="EN-US">Overview | ||
of RepVP123 | of RepVP123 | ||
plasmid</span><span | plasmid</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;">2</span></a></p> | + | style="color: windowtext; display: none; text-decoration: none;">2</span></a></p> |
- | <p class="MsoToc2"><a href="#_Toc275885922"><span lang="EN-US">Modularization: | + | <p class="MsoToc2"><a href="#_Toc275885922"><span |
+ | lang="EN-US">Modularization: | ||
Overview</span><span | Overview</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;">2</span></a></p> | + | style="color: windowtext; display: none; text-decoration: none;">2</span></a></p> |
- | <p class="MsoToc3"><a href="#_Toc275885923"><span lang="EN-US">Modularization: | + | <p class="MsoToc3"><a href="#_Toc275885923"><span |
+ | lang="EN-US">Modularization: | ||
Removing iGEM restriction sites and establishing loop insertion | Removing iGEM restriction sites and establishing loop insertion | ||
capability</span><span | capability</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></p> | + | style="color: windowtext; display: none; text-decoration: none;">3</span></a></p> |
- | <p class="MsoToc4"><a href="#_Toc275885924"><span lang="EN-US">Modifications | + | <p class="MsoToc4"><a href="#_Toc275885924"><span |
+ | lang="EN-US">Modifications | ||
in Rep</span><span | in Rep</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></p> | + | style="color: windowtext; display: none; text-decoration: none;">3</span></a></p> |
- | <p class="MsoToc4"><a href="#_Toc275885925"><span lang="EN-US">Modifications | + | <p class="MsoToc4"><a href="#_Toc275885925"><span |
+ | lang="EN-US">Modifications | ||
in | in | ||
VP123</span><span | VP123</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;">4</span></a></p> | + | style="color: windowtext; display: none; text-decoration: none;">4</span></a></p> |
- | <p class="MsoToc3"><a href="#_Toc275885926"><span lang="EN-US">Modularization: | + | <p class="MsoToc3"><a href="#_Toc275885926"><span |
+ | lang="EN-US">Modularization: | ||
Adapting pSB1C3 to loop insertions – pSB1C3_001</span><span | Adapting pSB1C3 to loop insertions – pSB1C3_001</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;">7</span></a></p> | + | style="color: windowtext; display: none; text-decoration: none;">7</span></a></p> |
- | <p class="MsoToc4"><a href="#_Toc275885927"><span lang="EN-US">Turning-off | + | <p class="MsoToc4"><a href="#_Toc275885927"><span |
+ | lang="EN-US">Turning-off | ||
natural | natural | ||
tropism: HSPG-knock-out</span><span | tropism: HSPG-knock-out</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;">9</span></a></p> | + | style="color: windowtext; display: none; text-decoration: none;">9</span></a></p> |
<p class="MsoToc2" style="margin-left: 0cm; text-indent: 0cm;"> </p> | <p class="MsoToc2" style="margin-left: 0cm; text-indent: 0cm;"> </p> | ||
<span | <span | ||
- | style="font-size: 11pt; line-height: 115%; font-family: "Calibri","sans-serif";" | + | style="font-size: 11pt; line-height: 115%; font-family: "Calibri","sans-serif";" |
- | lang="EN-US"><br style="page-break-before: always;" clear="all"> | + | lang="EN-US"><br style="page-break-before: always;" |
+ | clear="all"> | ||
</span> | </span> | ||
- | <h2><a name="_Toc275800680">Introduction to Modularization of vector | + | <h2><a name="_Toc275800680">Introduction to |
+ | Modularization of vector | ||
plasmid</a></h2> | plasmid</a></h2> | ||
<br> | <br> | ||
- | <p class="MsoNormal"><span lang="EN-US">Producing recombinant virus | + | <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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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="_Toc275797954"><span lang="EN-US"><span | + | name="_Toc275800681"></a><a name="_Toc275797954"><span |
- | style="font-family: "Times New Roman"; 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"><span |
- | lang="EN-US">Recombinant and Modular Vectorplasmid Carrying | + | style="font-family: "Times New Roman"; 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">Recombinant and Modular Vectorplasmid Carrying | ||
GOI</span></a></h3> | GOI</span></a></h3> | ||
- | <p class="MsoNormal"><span lang="EN-US">The iGEM team Freiburg_Bioware | + | <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 | ||
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<div align="center"> | <div align="center"> | ||
<table class="MsoTableGrid" | <table class="MsoTableGrid" | ||
- | style="border: medium none ; border-collapse: collapse;" border="0" | + | style="border: medium none ; border-collapse: collapse;" |
- | cellpadding="0" cellspacing="0"> | + | border="0" cellpadding="0" cellspacing="0"> |
- | <tbody> | + | <tbody> |
- | <tr style="height: 522.25pt;"> | + | <tr style="height: 522.25pt;"> |
- | <td style="padding: 0cm 5.4pt; width: 441.75pt; height: 522.25pt;" | + | <td |
- | valign="top" width="589"> | + | style="padding: 0cm 5.4pt; width: 441.75pt; height: 522.25pt;" |
- | <p class="MsoNormal" | + | valign="top" width="589"> |
- | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img | + | <p class="MsoNormal" |
- | id="Grafik 5" | + | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img |
- | src="https://static.igem.org/mediawiki/2010/a/ae/Freiburg10_Overview_BBa_Vectorplasmid.png" | + | id="Grafik 5" |
- | height="692" width="580"></p> | + | src="https://static.igem.org/mediawiki/2010/a/ae/Freiburg10_Overview_BBa_Vectorplasmid.png" |
- | <p class="MsoCaption" style="text-indent: 0cm;"><span lang="EN-US">Figure | + | height="692" width="580"></p> |
- | </span><span lang="EN-US">1</span><span lang="EN-US">: </span><span | + | <p class="MsoCaption" style="text-indent: 0cm;"><span |
- | style="font-weight: normal;" lang="EN-US">Overview of the theoretical | + | lang="EN-US">Figure |
+ | </span><span lang="EN-US">1</span><span | ||
+ | lang="EN-US">: </span><span | ||
+ | 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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these constructs can be found under ‘Arming: Killing the tumor’ and | these constructs can be found under ‘Arming: Killing the tumor’ and | ||
‘N-terminal fusion for Targeting’.</span></p> | ‘N-terminal fusion for Targeting’.</span></p> | ||
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | </tbody> | + | </tbody> |
</table> | </table> | ||
</div> | </div> | ||
- | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US"> </span></p> | + | <p class="MsoNormal" style="text-indent: 0cm;"><span |
- | <h3><a name="_Toc275800682"></a><a name="_Toc275797955"><span | + | lang="EN-US"> </span></p> |
- | lang="EN-US">Cloning | + | <h3><a name="_Toc275800682"></a><a |
+ | name="_Toc275797955"><span lang="EN-US">Cloning | ||
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 | ||
Line 224: | Line 269: | ||
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 | ||
Line 231: | Line 277: | ||
TK30 (BBa_K404109) | TK30 (BBa_K404109) | ||
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">BBa_K404112</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 | ||
‘Arming – | ‘Arming – | ||
Line 244: | Line 290: | ||
Cloning | Cloning | ||
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 |
- | 2</span><span lang="EN-US"> the schematic overview of the | + | lang="EN-US">Figure |
+ | 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 251: | Line 299: | ||
<div align="center"> | <div align="center"> | ||
<table class="MsoTableGrid" | <table class="MsoTableGrid" | ||
- | style="border: medium none ; border-collapse: collapse;" border="0" | + | style="border: medium none ; border-collapse: collapse;" |
- | cellpadding="0" cellspacing="0"> | + | border="0" cellpadding="0" cellspacing="0"> |
- | <tbody> | + | <tbody> |
- | <tr style="height: 26.15pt;"> | + | <tr style="height: 26.15pt;"> |
- | <td style="padding: 0cm 5.4pt; width: 340.15pt; height: 26.15pt;" | + | <td |
- | valign="top" width="454"> | + | style="padding: 0cm 5.4pt; width: 340.15pt; height: 26.15pt;" |
- | <p class="MsoNormal" | + | valign="top" width="454"> |
- | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img | + | <p class="MsoNormal" |
- | id="Grafik 2" | + | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img |
- | src="https://static.igem.org/mediawiki/2010/3/39/Freiburg10_Overview_Assembly_GOI.png" | + | id="Grafik 2" |
- | alt="Beschreibung: http://partsregistry.org/wiki/images/1/1c/Freiburg10_Vectorplasmid_cloning.png" | + | src="https://static.igem.org/mediawiki/2010/3/39/Freiburg10_Overview_Assembly_GOI.png" |
- | height="272" width="439"></p> | + | alt="Beschreibung: http://partsregistry.org/wiki/images/1/1c/Freiburg10_Vectorplasmid_cloning.png" |
- | <p class="MsoCaption" style="text-indent: 0cm;"><a | + | height="272" width="439"></p> |
- | name="_Ref275783119"><span lang="EN-US">Figure </span></a><span | + | <p class="MsoCaption" style="text-indent: 0cm;"><a |
- | lang="EN-US">3</span><span lang="EN-US">: </span><span | + | name="_Ref275783119"><span lang="EN-US">Figure </span></a><span |
- | style="font-weight: normal;" lang="EN-US">Assembly procedure for | + | lang="EN-US">3</span><span lang="EN-US">: |
+ | </span><span style="font-weight: normal;" | ||
+ | lang="EN-US">Assembly procedure for | ||
fusion of BioBricks and composite parts to a fully assembled and | fusion of BioBricks and composite parts to a fully assembled and | ||
functional plasmid coding for your gene of interest. This plasmid can | functional plasmid coding for your gene of interest. This plasmid can | ||
Line 272: | Line 322: | ||
assembly and encapsidating of the rAAV genome (your gene of interest) | assembly and encapsidating of the rAAV genome (your gene of interest) | ||
into the capsids.</span></p> | into the capsids.</span></p> | ||
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | </tbody> | + | </tbody> |
</table> | </table> | ||
</div> | </div> | ||
<p class="MsoNormal"><span lang="EN-US"> </span></p> | <p class="MsoNormal"><span lang="EN-US"> </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 286: | Line 337: | ||
which are | which are | ||
left ITR and CMV or phTERT promoter, respectively.</span></p> | left ITR and CMV or phTERT promoter, respectively.</span></p> | ||
- | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">As | + | <p class="MsoNormal" style="text-indent: 0cm;"><span |
- | shown in </span><span lang="EN-US">Figure 3</span><span lang="EN-US"> | + | lang="EN-US">As |
+ | 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="0" | + | style="border: medium none ; border-collapse: collapse;" |
- | cellpadding="0" cellspacing="0"> | + | border="0" cellpadding="0" cellspacing="0"> |
- | <tbody> | + | <tbody> |
- | <tr style="height: 23.95pt;"> | + | <tr style="height: 23.95pt;"> |
- | <td style="padding: 0cm 5.4pt; width: 349.9pt; height: 23.95pt;" | + | <td |
- | valign="top" width="467"> | + | style="padding: 0cm 5.4pt; width: 349.9pt; height: 23.95pt;" |
- | <p class="MsoNormal" | + | valign="top" width="467"> |
- | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img | + | <p class="MsoNormal" |
- | id="Grafik 74" | + | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img |
- | src="https://static.igem.org/mediawiki/2010/e/e3/Freiburg10_TheoreticalCloning_ITR_CMV_beta.png" | + | id="Grafik 74" |
- | height="530" width="452"></p> | + | src="https://static.igem.org/mediawiki/2010/e/e3/Freiburg10_TheoreticalCloning_ITR_CMV_beta.png" |
- | <p class="MsoCaption" style="text-indent: 0cm;"><a | + | height="530" width="452"></p> |
- | name="_Ref275783160"><span lang="EN-US">Figure </span></a><span | + | <p class="MsoCaption" style="text-indent: 0cm;"><a |
- | lang="EN-US">4</span><span lang="EN-US">:</span><span lang="EN-US"> </span><span | + | name="_Ref275783160"><span lang="EN-US">Figure </span></a><span |
- | style="font-weight: normal;" lang="EN-US">Theoretical cloning of the | + | lang="EN-US">4</span><span lang="EN-US">:</span><span |
- | composite part leftITR_CMV to the <i>beta-globin</i> intron BioBrick | + | lang="EN-US"> </span><span |
- | leading to the plasmid leftITR_CMV_<i>beta-globin</i> intron.</span></p> | + | style="font-weight: normal;" lang="EN-US">Theoretical |
- | </td> | + | cloning of the |
- | </tr> | + | composite part leftITR_CMV to the <i>beta-globin</i> |
- | </tbody> | + | intron BioBrick |
+ | leading to the plasmid leftITR_CMV_<i>beta-globin</i> | ||
+ | intron.</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
</table> | </table> | ||
</div> | </div> | ||
- | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US"> </span></p> | + | <p class="MsoNormal" style="text-indent: 0cm;"><span |
- | <p class="MsoNormal"><span lang="EN-US">The plasmids were digested with | + | lang="EN-US"> </span></p> |
+ | <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">) or SpeI and | + | lang="EN-US">BBa_K404107</span><span 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 gel in </span><span lang="EN-US">Figure 4</span><span | + | preparative gel in </span><span lang="EN-US">Figure |
- | lang="EN-US">, | + | 4</span><span lang="EN-US">, |
the expected bands could be detected under UV light and the extracted | the expected bands could be detected under UV light and the extracted | ||
DNA could | DNA could | ||
Line 331: | Line 393: | ||
<div align="center"> | <div align="center"> | ||
<table class="MsoTableGrid" | <table class="MsoTableGrid" | ||
- | style="border: medium none ; border-collapse: collapse;" border="0" | + | style="border: medium none ; border-collapse: collapse;" |
- | cellpadding="0" cellspacing="0"> | + | border="0" cellpadding="0" cellspacing="0"> |
- | <tbody> | + | <tbody> |
- | <tr style="height: 129.95pt;"> | + | <tr style="height: 129.95pt;"> |
- | <td style="padding: 0cm 5.4pt; width: 312.75pt; height: 129.95pt;" | + | <td |
- | valign="top" width="417"> | + | style="padding: 0cm 5.4pt; width: 312.75pt; height: 129.95pt;" |
- | <p class="MsoNormal" | + | valign="top" width="417"> |
- | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span | + | <p class="MsoNormal" |
- | lang="EN-US"> </span></p> | + | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span |
- | <p class="MsoNormal" | + | lang="EN-US"> </span></p> |
- | style="margin-bottom: 0.0001pt; text-align: right; text-indent: 0cm; line-height: normal; page-break-after: avoid;" | + | <p class="MsoNormal" |
- | align="right"><img id="Grafik 77" | + | style="margin-bottom: 0.0001pt; text-align: right; text-indent: 0cm; line-height: normal; page-break-after: avoid;" |
- | src="https://static.igem.org/mediawiki/2010/4/49/Freiburg10_Cloning_Intermediate_GOI.png" | + | align="right"><img id="Grafik 77" |
- | 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" | + | src="https://static.igem.org/mediawiki/2010/4/49/Freiburg10_Cloning_Intermediate_GOI.png" |
- | height="194" width="397"></p> | + | 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" |
- | <p class="MsoCaption" style="text-indent: 0cm;"><span lang="EN-US">Figure | + | height="194" width="397"></p> |
- | </span><span lang="EN-US">5</span><span lang="EN-US">: Assembly | + | <p class="MsoCaption" style="text-indent: 0cm;"><span |
+ | lang="EN-US">Figure | ||
+ | </span><span lang="EN-US">5</span><span | ||
+ | lang="EN-US">: Assembly | ||
intermediate in fusion of the vectorplasmids containing different | intermediate in fusion of the vectorplasmids containing different | ||
- | 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">BBa_K404107</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 362: | Line 429: | ||
Insert, pSB1C3_<i>beta-globin</i> intron; Vector pCMV, | Insert, pSB1C3_<i>beta-globin</i> intron; Vector pCMV, | ||
pSB1C3_leftITR_pCMV; Vector phTERT, pSB1C3_leftITR_phTERT.</span></p> | pSB1C3_leftITR_pCMV; Vector phTERT, pSB1C3_leftITR_phTERT.</span></p> | ||
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | </tbody> | + | </tbody> |
</table> | </table> | ||
</div> | </div> | ||
<p class="MsoNormal"><span lang="EN-US"> </span></p> | <p class="MsoNormal"><span lang="EN-US"> </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 381: | Line 450: | ||
leftITR_CMV_<i>beta-globin</i> | leftITR_CMV_<i>beta-globin</i> | ||
intron (BBa_K404117).</span></p> | intron (BBa_K404117).</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 390: | Line 460: | ||
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 |
+ | and | ||
pSB1C3_hGH_rightITR (BBa_K404116). The fragment obtained after | pSB1C3_hGH_rightITR (BBa_K404116). The fragment obtained after | ||
digestion on the | digestion on the | ||
Line 401: | Line 473: | ||
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">). </span></p> | + | lang="EN-US">BBa_K404124</span><span 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="0" | + | style="border: medium none ; border-collapse: collapse;" |
- | cellpadding="0" cellspacing="0"> | + | border="0" cellpadding="0" cellspacing="0"> |
- | <tbody> | + | <tbody> |
- | <tr style="height: 136.9pt;"> | + | <tr style="height: 136.9pt;"> |
- | <td style="padding: 0cm 5.4pt; width: 308.4pt; height: 136.9pt;" | + | <td |
- | valign="top" width="411"> | + | style="padding: 0cm 5.4pt; width: 308.4pt; height: 136.9pt;" |
- | <p class="MsoNormal" | + | valign="top" width="411"> |
- | style="margin-bottom: 0.0001pt; text-align: right; text-indent: 0cm; line-height: normal; page-break-after: avoid;" | + | <p class="MsoNormal" |
- | align="right"><img id="Grafik 80" | + | style="margin-bottom: 0.0001pt; text-align: right; text-indent: 0cm; line-height: normal; page-break-after: avoid;" |
- | src="https://static.igem.org/mediawiki/2010/9/93/Freiburg10_Cloning_Full_GOI.png" | + | align="right"><img id="Grafik 80" |
- | alt="Beschreibung: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\18.09_Cloning_Full_phTERT_mVenus.png" | + | src="https://static.igem.org/mediawiki/2010/9/93/Freiburg10_Cloning_Full_GOI.png" |
- | height="184" width="397"></p> | + | alt="Beschreibung: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\18.09_Cloning_Full_phTERT_mVenus.png" |
- | <p class="MsoCaption" style="text-indent: 0cm;"><a | + | height="184" width="397"></p> |
- | name="_Ref275784510"><span lang="EN-US">Figure </span></a><span | + | <p class="MsoCaption" style="text-indent: 0cm;"><a |
- | lang="EN-US">6</span><span style="font-weight: normal;" lang="EN-US">: | + | name="_Ref275784510"><span lang="EN-US">Figure </span></a><span |
- | </span><span lang="EN-US">Modularization of the assembled | + | lang="EN-US">6</span><span |
+ | style="font-weight: normal;" lang="EN-US">: | ||
+ | </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 | ||
of the composite pSB1C3_leftITR_phTERT_beta-globin intron_mVenus part | of the composite pSB1C3_leftITR_phTERT_beta-globin intron_mVenus part | ||
to the composite parts pSB1C3_hGH_rightITR was performed following the | to the composite parts pSB1C3_hGH_rightITR was performed following the | ||
Line 432: | Line 509: | ||
GeneRuler DNA ladder mix; Vector, pSB1C3_leftITR_phTERT_beta-globin | GeneRuler DNA ladder mix; Vector, pSB1C3_leftITR_phTERT_beta-globin | ||
intron_mVenus; Insert, pSB1C3_ pSB1C3_hGH_rightITR.</span></p> | intron_mVenus; Insert, pSB1C3_ pSB1C3_hGH_rightITR.</span></p> | ||
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | </tbody> | + | </tbody> |
</table> | </table> | ||
</div> | </div> | ||
- | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US"> </span></p> | + | <p class="MsoNormal" style="text-indent: 0cm;"><span |
- | <p class="MsoNormal"><span lang="EN-US">Since cloning does not confirm | + | lang="EN-US"> </span></p> |
+ | <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. |
+ | Assembled | ||
plasmids | plasmids | ||
have been cotransfected, using AAV-293 cells, which provide the stable | have been cotransfected, using AAV-293 cells, which provide the stable | ||
Line 459: | Line 539: | ||
visualization | visualization | ||
using fluorescent microscopy and flow cytometry analysis.</span></p> | using fluorescent microscopy and flow cytometry analysis.</span></p> | ||
- | <h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800683"></a><a | + | <h3 style="margin-left: 0cm; text-indent: 0cm;"><a |
- | name="_Toc275797956"><span lang="EN-US"><span | + | name="_Toc275800683"></a><a name="_Toc275797956"><span |
- | style="font-family: "Times New Roman"; 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"><span |
- | lang="EN-US">Testing functionality of Assembled Vectorplasmid</span></a></h3> | + | style="font-family: "Times New Roman"; 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 |
- | <h4><a name="_Toc275800684"></a><a name="_Toc275797957"><span | + | lang="EN-US">Testing functionality of Assembled Vectorplasmid</span></a></h3> |
- | lang="EN-US">Fluorescence | + | <h4><a name="_Toc275800684"></a><a |
+ | name="_Toc275797957"><span 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 472: | Line 554: | ||
transduced HT1080 cells and non-transduced cells could be easily | transduced HT1080 cells and non-transduced cells could be easily | ||
distinguished. | distinguished. | ||
- | In </span><span lang="EN-US">Figure 6</span><span lang="EN-US"> cells | + | In </span><span lang="EN-US">Figure 6</span><span |
+ | lang="EN-US"> cells | ||
were excited | were excited | ||
with 505nm and fluorescence emission at 536nm was detected. Therefore, | with 505nm and fluorescence emission at 536nm was detected. Therefore, | ||
Line 481: | Line 564: | ||
<div align="center"> | <div align="center"> | ||
<table class="MsoTableGrid" | <table class="MsoTableGrid" | ||
- | style="border: medium none ; border-collapse: collapse;" border="0" | + | style="border: medium none ; border-collapse: collapse;" |
- | cellpadding="0" cellspacing="0"> | + | border="0" cellpadding="0" cellspacing="0"> |
- | <tbody> | + | <tbody> |
- | <tr> | + | <tr> |
- | <td style="padding: 0cm 5.4pt; width: 250.7pt;" valign="top" | + | <td style="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 |
- | lang="EN-US">A</span></p> | + | lang="EN-US">A</span></p> |
- | <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 id="Grafik 18" |
- | src="Freiburg10_Modularization_GOI_forWiki-Dateien/image006.jpg" | + | src="Freiburg10_Modularization_GOI_forWiki-Dateien/image006.jpg" |
- | alt="Beschreibung: Freiburg10_2Transd30µg_unverd_2_(c1).JPG (1388×1040)" | + | alt="Beschreibung: Freiburg10_2Transd30µg_unverd_2_(c1).JPG (1388×1040)" |
- | height="218" width="264"></p> | + | height="218" width="264"></p> |
- | </td> | + | </td> |
- | <td style="padding: 0cm 5.4pt; width: 230.4pt;" valign="top" | + | <td style="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 |
- | lang="EN-US">B</span></p> | + | lang="EN-US">B</span></p> |
- | <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 id="Grafik 19" |
- | src="Freiburg10_Modularization_GOI_forWiki-Dateien/image007.jpg" | + | src="Freiburg10_Modularization_GOI_forWiki-Dateien/image007.jpg" |
- | alt="Beschreibung: https://static.igem.org/mediawiki/2010/f/f1/Freiburg10_2Transd30%C2%B5g_unverd_%28c1%29.JPG" | + | alt="Beschreibung: https://static.igem.org/mediawiki/2010/f/f1/Freiburg10_2Transd30%C2%B5g_unverd_%28c1%29.JPG" |
- | height="220" width="242"></p> | + | height="220" width="242"></p> |
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | <tr> | + | <tr> |
- | <td style="padding: 0cm 5.4pt; width: 250.7pt;" valign="top" | + | <td style="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 |
- | lang="EN-US">C</span></p> | + | lang="EN-US">C</span></p> |
- | <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 id="Grafik 16" |
- | src="Freiburg10_Modularization_GOI_forWiki-Dateien/image008.jpg" | + | src="Freiburg10_Modularization_GOI_forWiki-Dateien/image008.jpg" |
- | alt="Beschreibung: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg" | + | alt="Beschreibung: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg" |
- | height="195" width="258"></p> | + | height="195" width="258"></p> |
- | </td> | + | </td> |
- | <td style="padding: 0cm 5.4pt; width: 230.4pt;" valign="top" | + | <td style="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 |
- | lang="EN-US">D</span></p> | + | lang="EN-US">D</span></p> |
- | <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 id="Grafik 17" |
- | src="Freiburg10_Modularization_GOI_forWiki-Dateien/image009.jpg" | + | src="Freiburg10_Modularization_GOI_forWiki-Dateien/image009.jpg" |
- | alt="Beschreibung: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg" | + | alt="Beschreibung: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg" |
- | height="194" width="257"></p> | + | height="194" width="257"></p> |
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | <tr> | + | <tr> |
- | <td colspan="2" style="padding: 0cm 5.4pt; width: 481.1pt;" | + | <td colspan="2" |
- | valign="top" width="641"> | + | style="padding: 0cm 5.4pt; width: 481.1pt;" valign="top" |
- | <p class="MsoCaption" style="text-indent: 0cm;"><a | + | width="641"> |
- | name="_Ref275784524"><span lang="EN-US">Figure </span></a><span | + | <p class="MsoCaption" style="text-indent: 0cm;"><a |
- | lang="EN-US">7</span><span lang="EN-US">: </span><span | + | name="_Ref275784524"><span lang="EN-US">Figure </span></a><span |
- | style="font-weight: normal;" lang="EN-US">Fluorescence microscopy | + | lang="EN-US">7</span><span lang="EN-US">: |
+ | </span><span style="font-weight: normal;" | ||
+ | lang="EN-US">Fluorescence microscopy | ||
(Exciatation: 505nm, Emission: 536nm) was performed for detection of | (Exciatation: 505nm, Emission: 536nm) was performed for detection of | ||
transduced cell expression mVenus. A:Cells detected in bright field | transduced cell expression mVenus. A:Cells detected in bright field | ||
picture B: Detection of mVenus expression can be observed.</span></p> | picture B: Detection of mVenus expression can be observed.</span></p> | ||
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | </tbody> | + | </tbody> |
</table> | </table> | ||
</div> | </div> | ||
<p class="MsoNormal"><span lang="EN-US"> </span></p> | <p class="MsoNormal"><span lang="EN-US"> </span></p> | ||
- | <h4><a name="_Toc275800685"></a><a name="_Toc275797958"><span | + | <h4><a name="_Toc275800685"></a><a |
- | lang="EN-US">Analysis | + | name="_Toc275797958"><span 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> | ||
- | <h5><a name="_Toc275800686"></a><a name="_Toc275797959"><span | + | <h5><a name="_Toc275800686"></a><a |
- | lang="EN-US">Influence | + | name="_Toc275797959"><span 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 569: | Line 657: | ||
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 581: | Line 670: | ||
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 | ||
Line 595: | Line 685: | ||
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"> confirming the expected results | + | lang="EN-US">Figure 8</span><span 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 604: | Line 695: | ||
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; text-align: left; margin-left: auto; margin-right: auto;" | + | style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;" |
- | border="0" cellpadding="0" cellspacing="0"> | + | border="0" cellpadding="0" cellspacing="0"> |
- | <tbody> | + | <tbody> |
- | <tr> | + | <tr> |
- | <td | + | <td |
- | style="padding: 0cm 5.4pt; vertical-align: top; width: 481.1pt;"> | + | style="padding: 0cm 5.4pt; vertical-align: top; width: 481.1pt;"> |
- | <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 hGH termination signal</span></b></p> | + | lang="EN-US">Vectorplasmid lacking hGH termination signal</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 30" | + | id="Grafik 30" |
- | src="https://static.igem.org/mediawiki/2010/c/c7/Freiburg10_FACS_woHGH.png" | + | 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 style="padding: 0cm 5.4pt; width: 481.1pt;" valign="top" | + | <td style="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">Vectorplasmid containing hGH terminator signal</span></b></p> | + | lang="EN-US">Vectorplasmid containing hGH terminator signal</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 2049" | + | id="Grafik 2049" |
- | src="https://static.igem.org/mediawiki/2010/7/70/Freiburg10_FACS_withHGH.png" | + | src="https://static.igem.org/mediawiki/2010/7/70/Freiburg10_FACS_withHGH.png" |
- | height="410" width="635"></p> | + | height="410" width="635"></p> |
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | <tr> | + | <tr> |
- | <td style="padding: 0cm 5.4pt; width: 481.1pt;" valign="top" | + | <td style="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 |
- | name="_Ref275784539"><b><span lang="EN-US">Figure </span></b></a><b><span | + | name="_Ref275784539"><b><span lang="EN-US">Figure |
- | lang="EN-US">8</span></b><b><span lang="EN-US">:</span></b><span | + | </span></b></a><b><span lang="EN-US">8</span></b><b><span |
- | lang="EN-US"> <b>Flow cytometry analysis of vectorplasmids with and | + | lang="EN-US">:</span></b><span lang="EN-US"> |
- | without hGH terminator.</b> A: Gating non transduced cells (control); | + | <b>Flow cytometry analysis of vectorplasmids with and |
+ | 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 648: | Line 741: | ||
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: "Cambria Math","serif";" | (R2 </span><span style="font-family: "Cambria Math","serif";" | ||
- | 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 | (BBa_K404127), pHelper, pRC). D: Transduced cells plotted against | ||
Line 656: | Line 750: | ||
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: "Cambria Math","serif";" | (R2 </span><span style="font-family: "Cambria Math","serif";" | ||
- | 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 (BBa_K404119), pHelper, | ||
Line 663: | Line 758: | ||
non-transduced (red) and transduced (green) cells applied against | non-transduced (red) and transduced (green) cells applied against | ||
mVenus.</span></p> | mVenus.</span></p> | ||
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | </tbody> | + | </tbody> |
</table> | </table> | ||
<p class="MsoNormal"><span lang="EN-US"> </span></p> | <p class="MsoNormal"><span 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="0" | + | style="border: medium none ; border-collapse: collapse;" |
- | cellpadding="0" cellspacing="0"> | + | border="0" cellpadding="0" cellspacing="0"> |
- | <tbody> | + | <tbody> |
- | <tr style="height: 319.2pt;"> | + | <tr style="height: 319.2pt;"> |
- | <td style="padding: 0cm 5.4pt; width: 351.1pt; height: 319.2pt;" | + | <td |
- | valign="top" width="468"> | + | style="padding: 0cm 5.4pt; width: 351.1pt; height: 319.2pt;" |
- | <p class="MsoNormal" | + | valign="top" width="468"> |
- | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span | + | <p class="MsoNormal" |
- | lang="EN-US"> </span></p> | + | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span |
- | <p class="MsoNormal" | + | lang="EN-US"> </span></p> |
- | style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;" | + | <p class="MsoNormal" |
- | align="center"><img id="Diagramm 3" | + | style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;" |
- | src="https://static.igem.org/mediawiki/2010/9/94/Freiburg10_Diagram_hGH.png" | + | align="center"><img id="Diagramm 3" |
- | height="355" width="473"></p> | + | src="https://static.igem.org/mediawiki/2010/9/94/Freiburg10_Diagram_hGH.png" |
- | <p class="MsoCaption" style="text-indent: 0cm;"><a | + | height="355" width="473"></p> |
- | name="_Ref275784545"><span lang="EN-US">Figure </span></a><span | + | <p class="MsoCaption" style="text-indent: 0cm;"><a |
- | lang="EN-US">9</span><span lang="EN-US">: Flow cytometry analysis of | + | name="_Ref275784545"><span lang="EN-US">Figure </span></a><span |
+ | 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 |
+ | expression of viral | ||
genomes was determined by flow cytomery 24 hour post infection. | 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 | ||
Line 695: | Line 793: | ||
recombinant viral vectors engineered by using components of the Virus | recombinant viral vectors engineered by using components of the Virus | ||
Construction Kit.</span></p> | Construction Kit.</span></p> | ||
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | </tbody> | + | </tbody> |
</table> | </table> | ||
</div> | </div> | ||
<p class="MsoCaption"><span lang="EN-US"> </span></p> | <p class="MsoCaption"><span lang="EN-US"> </span></p> | ||
- | <h5><a name="_Toc275800687"></a><a name="_Toc275797960"><span | + | <h5><a name="_Toc275800687"></a><a |
- | lang="EN-US">Influence | + | name="_Toc275797960"><span 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 714: | Line 816: | ||
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 | ||
- | lang="EN-US">(Nott et al. 2003)</span><span lang="EN-US">. Analysis | + | lang="EN-US">(Nott et al. 2003)</span><span |
+ | lang="EN-US">. Analysis | ||
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 734: | Line 841: | ||
lacking the | lacking the | ||
beta-globin intron is minimal. Since packaging efficiency of the AAV-2 | beta-globin intron is minimal. Since packaging efficiency of the AAV-2 | ||
- | decreases with increasing sizes of the insert </span><span lang="EN-US">(Dong | + | decreases with increasing sizes of the insert </span><span |
- | et al. 1996)</span><span lang="EN-US">, the iGEM team | + | lang="EN-US">(Dong |
- | Freiburg_Bioware suggests using the <i>beta-globin </i>intron in | + | et al. 1996)</span><span lang="EN-US">, the iGEM |
+ | 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"> | <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="0" cellpadding="0" cellspacing="0" width="654"> | + | border="0" cellpadding="0" cellspacing="0" |
- | <tbody> | + | width="654"> |
- | <tr style="height: 2.5pt;"> | + | <tbody> |
- | <td style="padding: 0cm 5.4pt; width: 490.75pt; height: 2.5pt;" | + | <tr style="height: 2.5pt;"> |
- | valign="top" width="654"> | + | <td style="padding: 0cm 5.4pt; width: 490.75pt; height: 2.5pt;" |
- | <p class="MsoNormal" | + | valign="top" width="654"> |
- | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span | + | <p class="MsoNormal" |
- | lang="EN-US">Vectorplasmid lacking <i>beta-globin</i> intron</span></b></p> | + | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span |
- | <p class="MsoNormal" | + | lang="EN-US">Vectorplasmid lacking <i>beta-globin</i> |
- | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img | + | intron</span></b></p> |
- | id="Grafik 55" | + | <p class="MsoNormal" |
- | src="https://static.igem.org/mediawiki/2010/7/7c/Freiburg10_FACS_betaglobin.png" | + | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img |
- | height="412" width="640"></p> | + | id="Grafik 55" |
- | </td> | + | src="https://static.igem.org/mediawiki/2010/7/7c/Freiburg10_FACS_betaglobin.png" |
- | </tr> | + | height="412" width="640"></p> |
- | <tr style="height: 106.5pt;"> | + | </td> |
- | <td style="padding: 0cm 5.4pt; width: 490.75pt; height: 106.5pt;" | + | </tr> |
- | valign="top" width="654"> | + | <tr style="height: 106.5pt;"> |
- | <p class="MsoNormal" | + | <td |
- | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span | + | style="padding: 0cm 5.4pt; width: 490.75pt; height: 106.5pt;" |
- | lang="EN-US">Vectorplasmid containing <i>beta-globin</i> intron</span></b></p> | + | valign="top" width="654"> |
- | <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;"><b><span |
- | id="Grafik 63" | + | lang="EN-US">Vectorplasmid containing <i>beta-globin</i> |
- | src="https://static.igem.org/mediawiki/2010/8/83/Freiburg10_FACS_withbetaglobin.png" | + | intron</span></b></p> |
- | height="410" width="635"></p> | + | <p class="MsoNormal" |
- | </td> | + | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img |
- | </tr> | + | id="Grafik 63" |
- | <tr style="height: 106.5pt;"> | + | src="https://static.igem.org/mediawiki/2010/8/83/Freiburg10_FACS_withbetaglobin.png" |
- | <td style="padding: 0cm 5.4pt; width: 490.75pt; height: 106.5pt;" | + | height="410" width="635"></p> |
- | valign="top" width="654"> | + | </td> |
- | <p class="MsoCaption" style="text-indent: 0cm;"><a | + | </tr> |
- | name="_Ref275784803"><span lang="EN-US">Figure </span></a><span | + | <tr style="height: 106.5pt;"> |
- | lang="EN-US">10</span><span lang="EN-US">: Flow cytometry analysis of | + | <td |
- | vectorplasmids with and without <i>beta-globin</i> intron. A</span><span | + | style="padding: 0cm 5.4pt; width: 490.75pt; height: 106.5pt;" |
- | style="font-weight: normal;" lang="EN-US">: Gating non transduced | + | valign="top" width="654"> |
+ | <p class="MsoCaption" style="text-indent: 0cm;"><a | ||
+ | name="_Ref275784803"><span lang="EN-US">Figure </span></a><span | ||
+ | lang="EN-US">10</span><span lang="EN-US">: | ||
+ | Flow cytometry analysis of | ||
+ | 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 |
- | style="font-weight: normal;" lang="EN-US">: Non transduced cells | + | lang="EN-US">B</span><span |
+ | 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 | ||
- | lang="EN-US">C</span><span style="font-weight: normal;" lang="EN-US">: | + | lang="EN-US">C</span><span |
+ | style="font-weight: normal;" lang="EN-US">: | ||
Gating transduced cells (R2 </span><span | Gating transduced cells (R2 </span><span | ||
- | style="font-family: "Cambria Math","serif"; font-weight: normal;" | + | style="font-family: "Cambria Math","serif"; font-weight: normal;" |
- | lang="EN-US">≙</span><span style="font-weight: normal;" lang="EN-US">R14) | + | lang="EN-US">≙</span><span |
- | (used plasmids for transfection: GOI: </span><span lang="EN-US">pSB1C3_lITR_CMV_mVenus_hGH_rITR | + | style="font-weight: normal;" lang="EN-US">R14) |
- | (BBa_K404128)</span><span style="font-weight: normal;" lang="EN-US">, | + | (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">, | ||
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 |
- | Gating non-transduced cells (control). </span><span lang="EN-US">G</span><span | + | style="font-weight: normal;" lang="EN-US">: |
- | style="font-weight: normal;" lang="EN-US">: Non-transduced cells | + | Gating non-transduced cells (control). </span><span |
+ | lang="EN-US">G</span><span | ||
+ | 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: "Cambria Math","serif"; font-weight: normal;" | + | style="font-family: "Cambria Math","serif"; 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 | + | (BBa_K404119)</span><span style="font-weight: normal;" |
- | lang="EN-US">I</span><span style="font-weight: normal;" lang="EN-US">: | + | 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 |
- | style="font-weight: normal;" lang="EN-US">: Overlay of non-transduced | + | lang="EN-US">J</span><span |
+ | 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> |
- | </tr> | + | </tr> |
- | </tbody> | + | </tbody> |
</table> | </table> | ||
</div> | </div> | ||
Line 820: | Line 954: | ||
<div align="center"> | <div align="center"> | ||
<table class="MsoTableGrid" | <table class="MsoTableGrid" | ||
- | style="border: medium none ; width: 306.65pt; border-collapse: collapse;" | + | style="border: medium none ; width: 306.65pt; border-collapse: collapse;" |
- | border="0" cellpadding="0" cellspacing="0" width="409"> | + | border="0" cellpadding="0" cellspacing="0" |
- | <tbody> | + | width="409"> |
- | <tr style="height: 87.3pt;"> | + | <tbody> |
- | <td style="padding: 0cm 5.4pt; width: 306.65pt; height: 87.3pt;" | + | <tr style="height: 87.3pt;"> |
- | valign="top" width="409"> | + | <td |
- | <p class="MsoNormal" | + | style="padding: 0cm 5.4pt; width: 306.65pt; height: 87.3pt;" |
- | style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;" | + | valign="top" width="409"> |
- | align="center"><img id="Diagramm 57" | + | <p class="MsoNormal" |
- | src="https://static.igem.org/mediawiki/2010/2/26/Freiburg10_Diagram_betaglobin.png.png" | + | style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;" |
- | height="332" width="450"></p> | + | align="center"><img id="Diagramm 57" |
- | <p class="MsoCaption" style="text-indent: 0cm;"><a | + | src="https://static.igem.org/mediawiki/2010/2/26/Freiburg10_Diagram_betaglobin.png.png" |
- | name="_Ref275784805"><span lang="EN-US">Figure </span></a><span | + | height="332" width="450"></p> |
- | lang="EN-US">11</span><span lang="EN-US">: Flow cytometry analysis of | + | <p class="MsoCaption" style="text-indent: 0cm;"><a |
+ | name="_Ref275784805"><span lang="EN-US">Figure </span></a><span | ||
+ | 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 | ||
Line 841: | Line 979: | ||
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> | ||
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | </tbody> | + | </tbody> |
</table> | </table> | ||
</div> | </div> | ||
- | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US"> </span></p> | + | <p class="MsoNormal" style="text-indent: 0cm;"><span |
- | <h5><a name="_Toc275800688"></a><a name="_Toc275797961"><span | + | lang="EN-US"> </span></p> |
- | lang="EN-US">Functionality | + | <h5><a name="_Toc275800688"></a><a |
+ | name="_Toc275797961"><span lang="EN-US">Functionality | ||
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 | + | <p class="MsoNormal"><span lang="EN-US">After |
+ | assembly of plasmids | ||
containing all required | containing all required | ||
elements (see </span><span lang="EN-US">Figure 1</span><span | elements (see </span><span lang="EN-US">Figure 1</span><span | ||
- | lang="EN-US">), | + | lang="EN-US">), |
functionality was tested in cell culture. AAV-293 cells stably | functionality was tested in cell culture. AAV-293 cells stably | ||
expressing E1A | expressing E1A | ||
Line 864: | Line 1,004: | ||
encapsidating the | encapsidating the | ||
gene of interest mVenus (BBa_I757008).</span></p> | gene of interest mVenus (BBa_I757008).</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 | ||
2010 | 2010 | ||
compared the standard-plasmid containing a subcloned mVenus | compared the standard-plasmid containing a subcloned mVenus | ||
Line 872: | Line 1,013: | ||
(pSB1C3_mVenus: BBa_K404119). Fluorescence expression data obtained by | (pSB1C3_mVenus: BBa_K404119). Fluorescence expression data obtained by | ||
flow | flow | ||
- | cytometry analysis are shown in </span><span lang="EN-US">Figure 11</span><span | + | cytometry analysis are shown in </span><span lang="EN-US">Figure |
- | lang="EN-US"> and </span><span lang="EN-US">Figure 12</span><span | + | 11</span><span lang="EN-US"> and </span><span |
- | lang="EN-US">. | + | lang="EN-US">Figure 12</span><span lang="EN-US">. |
Comparing mVenus expression of the standard plasmid and the modified, | Comparing mVenus expression of the standard plasmid and the modified, | ||
assembled | assembled | ||
Line 882: | Line 1,023: | ||
<div align="center"> | <div align="center"> | ||
<table class="MsoTableGrid" | <table class="MsoTableGrid" | ||
- | style="border: medium none ; border-collapse: collapse;" border="0" | + | style="border: medium none ; border-collapse: collapse;" |
- | cellpadding="0" cellspacing="0"> | + | border="0" cellpadding="0" cellspacing="0"> |
- | <tbody> | + | <tbody> |
- | <tr> | + | <tr> |
- | <td style="padding: 0cm 5.4pt; width: 481.1pt;" valign="top" | + | <td style="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> | (BBa_K404119)</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="https://static.igem.org/mediawiki/2010/9/9e/Freiburg10_FACS_FULL_pSB1c3_mVenus.png" | + | src="https://static.igem.org/mediawiki/2010/9/9e/Freiburg10_FACS_FULL_pSB1c3_mVenus.png" |
- | height="409" width="634"></p> | + | height="409" width="634"></p> |
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | <tr> | + | <tr> |
- | <td style="padding: 0cm 5.4pt; width: 481.1pt;" valign="top" | + | <td style="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 (Stratagene)</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" | + | id="Grafik 89" |
- | src="https://static.igem.org/mediawiki/2010/a/ad/Freiburg10_FACS_FULL_pAAV_mVenus.png" | + | 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 style="padding: 0cm 5.4pt; width: 481.1pt;" valign="top" | + | <td style="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 | reassembled vectorplasmid (BBa_K404119) compared to standard plasmid | ||
- | provided by Stratagene. A</span><span style="font-weight: normal;" | + | provided by Stratagene. A</span><span |
- | lang="EN-US">: Gating non transduced cells (control); subcellular | + | style="font-weight: normal;" lang="EN-US">: Gating |
+ | non transduced cells (control); subcellular | ||
debris and clumps can be distinguished from single cells by size, | debris and clumps can be distinguished from single cells by size, | ||
estimated forward scatter (FS Lin) and granularity, estimated side | estimated forward scatter (FS Lin) and granularity, estimated side | ||
Line 926: | Line 1,069: | ||
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 | (R2 </span><span | ||
- | style="font-family: "Cambria Math","serif"; font-weight: normal;" | + | style="font-family: "Cambria Math","serif"; font-weight: normal;" |
- | lang="EN-US">≙</span><span style="font-weight: normal;" lang="EN-US">R14) | + | lang="EN-US">≙</span><span |
- | (used plasmids for transfection: pGOI: </span><span lang="EN-US">pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR | + | style="font-weight: normal;" lang="EN-US">R14) |
+ | (used plasmids for transfection: pGOI: </span><span | ||
+ | lang="EN-US">pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR | ||
(pSB1C3_mVenus: </span><span lang="EN-US">BBa_K404119</span><span | (pSB1C3_mVenus: </span><span lang="EN-US">BBa_K404119</span><span | ||
- | style="color: rgb(0, 176, 80); font-weight: normal;" lang="EN-US">)</span><span | + | style="color: rgb(0, 176, 80); font-weight: normal;" |
- | style="font-weight: normal;" lang="EN-US">, pHelper, pRC. </span><span | + | lang="EN-US">)</span><span |
- | lang="EN-US">D</span><span style="font-weight: normal;" lang="EN-US">: | + | style="font-weight: normal;" lang="EN-US">, pHelper, |
+ | pRC. </span><span lang="EN-US">D</span><span | ||
+ | style="font-weight: normal;" lang="EN-US">: | ||
Transduced cells plotted against mVenus, R10 comprised transduced | Transduced cells plotted against mVenus, R10 comprised transduced | ||
- | cells, by detecting mVenus expression. </span><span lang="EN-US">E</span><span | + | cells, by detecting mVenus expression. </span><span |
- | style="font-weight: normal;" lang="EN-US">: Overlay of non-transduced | + | lang="EN-US">E</span><span |
+ | style="font-weight: normal;" lang="EN-US">: Overlay | ||
+ | of non-transduced | ||
(red) and transduced (green). </span><span lang="EN-US">F</span><span | (red) and transduced (green). </span><span lang="EN-US">F</span><span | ||
- | style="font-weight: normal;" lang="EN-US">: Gating non transduced | + | style="font-weight: normal;" lang="EN-US">: Gating |
+ | non transduced | ||
cells (control). </span><span lang="EN-US">G</span><span | 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 | ||
plotted against mVenus (R5). </span><span lang="EN-US">H</span><span | plotted 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 | ||
(R14 </span><span | (R14 </span><span | ||
- | style="font-family: "Cambria Math","serif"; font-weight: normal;" | + | style="font-family: "Cambria Math","serif"; font-weight: normal;" |
- | lang="EN-US">≙</span><span style="font-weight: normal;" lang="EN-US">R2) | + | lang="EN-US">≙</span><span |
+ | style="font-weight: normal;" lang="EN-US">R2) | ||
(used plasmids for transfection: pGOI: pAAV_mVenus, pHelper). </span><span | (used plasmids for transfection: pGOI: pAAV_mVenus, pHelper). </span><span | ||
- | lang="EN-US">I</span><span style="font-weight: normal;" lang="EN-US">: | + | lang="EN-US">I</span><span |
+ | style="font-weight: normal;" lang="EN-US">: | ||
Transduced cells plotted against mVenus, R10 comprised transduced | Transduced cells plotted 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"> |
- | style="font-weight: normal;" lang="EN-US">: Overlay of non-transduced | + | J</span><span style="font-weight: normal;" lang="EN-US">: |
- | (red) and transduced (green) cells plotted against mVenus fluorescence. </span></p> | + | Overlay of non-transduced |
- | </td> | + | (red) and transduced (green) cells plotted against mVenus fluorescence. |
- | </tr> | + | </span></p> |
- | </tbody> | + | </td> |
+ | </tr> | ||
+ | </tbody> | ||
</table> | </table> | ||
</div> | </div> | ||
- | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US"> </span></p> | + | <p class="MsoNormal" style="text-indent: 0cm;"><span |
+ | lang="EN-US"> </span></p> | ||
<p class="MsoNormal"><span lang="EN-US"> </span></p> | <p class="MsoNormal"><span 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="0" | + | style="border: medium none ; border-collapse: collapse;" |
- | cellpadding="0" cellspacing="0"> | + | border="0" cellpadding="0" cellspacing="0"> |
- | <tbody> | + | <tbody> |
- | <tr> | + | <tr> |
- | <td style="padding: 0cm 5.4pt; width: 481.1pt;" valign="top" | + | <td style="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 |
- | lang="EN-US"> </span></p> | + | lang="EN-US"> </span></p> |
- | <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 90" | + | align="center"><img id="Diagramm 90" |
- | src="https://static.igem.org/mediawiki/2010/9/90/Freiburg10_Diagram_FULL_GOI.png" | + | src="https://static.igem.org/mediawiki/2010/9/90/Freiburg10_Diagram_FULL_GOI.png" |
- | height="396" 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">: |
+ | Flow cytometry analysis of | ||
reassembled vectorplasmid (BBa_K404119) compared to standard plasmid | reassembled vectorplasmid (BBa_K404119) compared to standard plasmid | ||
- | provided by Stratagene. </span><span style="font-weight: normal;" | + | provided by Stratagene. </span><span |
- | lang="EN-US">Fluorescence of the standard plasmid pAAV_mVenus | + | style="font-weight: normal;" lang="EN-US">Fluorescence |
+ | of the standard plasmid pAAV_mVenus | ||
(Stratagene) and the recombinant pSB1C3_mVenus (BBa_K404119) construct | (Stratagene) and the recombinant pSB1C3_mVenus (BBa_K404119) construct | ||
was measured. As demonstrated mVenus expression is enhanced in the | was measured. As demonstrated mVenus expression is enhanced in the | ||
assembled plasmid (pSB1C3_mVenus) compared to the standard pAAV_mVenus | assembled plasmid (pSB1C3_mVenus) compared to the standard pAAV_mVenus | ||
construct.</span></p> | construct.</span></p> | ||
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | </tbody> | + | </tbody> |
</table> | </table> | ||
</div> | </div> | ||
- | <h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800689"><span | + | <h3 style="margin-left: 0cm; text-indent: 0cm;"><a |
- | lang="EN-US"><span | + | name="_Toc275800689"><span lang="EN-US"><span |
- | style="font-family: "Times New Roman"; 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: "Times New Roman"; 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 1,022: | Line 1,182: | ||
the | the | ||
tumor’. </span></p> | tumor’. </span></p> | ||
- | <h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800690"><span | + | <h3 style="margin-left: 0cm; text-indent: 0cm;"><a |
- | lang="EN-US"><span | + | name="_Toc275800690"><span lang="EN-US"><span |
- | style="font-family: "Times New Roman"; 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: "Times New Roman"; 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">References</span></a></h3> | + | lang="EN-US">References</span></a></h3> |
<p style="text-indent: 36pt;"><span | <p style="text-indent: 36pt;"><span | ||
- | style="font-size: 10pt; font-family: "Calibri","sans-serif";" | + | style="font-size: 10pt; font-family: "Calibri","sans-serif";" |
- | lang="EN-US">Danckwardt, S., Hentze, M.W. & Kulozik, | + | lang="EN-US">Danckwardt, S., Hentze, M.W. & Kulozik, |
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> | ||
<p style="text-indent: 36pt;"><span | <p style="text-indent: 36pt;"><span | ||
- | style="font-size: 10pt; font-family: "Calibri","sans-serif";" | + | style="font-size: 10pt; font-family: "Calibri","sans-serif";" |
- | lang="EN-US">Dong, J.Y., Fan, P.D. & Frizzell, R.a., | + | lang="EN-US">Dong, J.Y., Fan, P.D. & 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> | ||
<p style="text-indent: 36pt;"><span | <p style="text-indent: 36pt;"><span | ||
- | style="font-size: 10pt; font-family: "Calibri","sans-serif";" | + | style="font-size: 10pt; font-family: "Calibri","sans-serif";" |
- | 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 | ||
- | style="font-size: 10pt; font-family: "Calibri","sans-serif";" | + | style="font-size: 10pt; font-family: "Calibri","sans-serif";" |
- | lang="EN-US">Nott, A., Meislin, S.H. & Moore, M.J., | + | lang="EN-US">Nott, A., Meislin, S.H. & Moore, M.J., |
2003. A quantitative analysis of intron effects on mammalian gene | 2003. A quantitative analysis of intron effects on mammalian gene | ||
expression. <i>RNA | expression. <i>RNA | ||
Line 1,056: | Line 1,219: | ||
<p style="text-indent: 36pt;"><span lang="EN-US"> </span></p> | <p style="text-indent: 36pt;"><span lang="EN-US"> </span></p> | ||
<p class="MsoNormal" | <p class="MsoNormal" | ||
- | style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;" | + | style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;" |
- | align="left"><span lang="EN-US"> </span></p> | + | align="left"><span lang="EN-US"> </span></p> |
- | <h2 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275885921"></a><a | + | <h2 style="margin-left: 0cm; text-indent: 0cm;"><a |
- | name="_Toc275817880"><span lang="EN-US">Overview of RepVP123 plasmid</span></a></h2> | + | name="_Toc275885921"></a><a name="_Toc275817880"><span |
- | <h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275885922"></a><a | + | lang="EN-US">Overview of RepVP123 plasmid</span></a></h2> |
- | name="_Toc275817881"><span lang="EN-US">Modularization: Overview</span></a></h3> | + | <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" | <p class="MsoNormal" | ||
- | style="text-indent: 0cm; line-height: 150%; page-break-after: avoid;"><span | + | style="text-indent: 0cm; line-height: 150%; page-break-after: avoid;"><span |
- | lang="EN-US">In our terminology the term “RepVP123” | + | lang="EN-US">In our terminology the term “RepVP123” |
encompasses the | encompasses the | ||
whole AAV2 genome excluding the ITRs. The <i>rep</i> locus | whole AAV2 genome excluding the ITRs. The <i>rep</i> locus | ||
Line 1,083: | Line 1,248: | ||
loops 453 | loops 453 | ||
and 587 a total of twelve mutations within RepVP123 (see </span><span | and 587 a total of twelve mutations within RepVP123 (see </span><span | ||
- | lang="EN-US">Figure 1</span><span lang="EN-US">) | + | lang="EN-US">Figure 1</span><span lang="EN-US">) |
and additionally two mutations | and additionally two mutations | ||
within the pSB1C3 backbone were performed either by Site-Directed | within the pSB1C3 backbone were performed either by Site-Directed | ||
Line 1,093: | Line 1,258: | ||
variant of this backbone, pSB1C3_001.</span></p> | variant of this backbone, pSB1C3_001.</span></p> | ||
<table class="MsoTableGrid" | <table class="MsoTableGrid" | ||
- | style="border: medium none ; border-collapse: collapse; width: 636px; height: 523px; text-align: left; margin-left: auto; margin-right: auto;" | + | 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"> | + | border="0" cellpadding="0" cellspacing="0"> |
- | <tbody> | + | <tbody> |
- | <tr> | + | <tr> |
- | <td | + | <td |
- | style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 462.5pt;"> | + | style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 462.5pt;"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="text-align: center; text-indent: 0cm; line-height: 150%; page-break-after: avoid;" | + | style="text-align: center; text-indent: 0cm; line-height: 150%; page-break-after: avoid;" |
- | align="center"><img style="width: 605px; height: 451px;" | + | align="center"><img style="width: 605px; height: 451px;" |
- | id="Picture 17" | + | id="Picture 17" |
- | src="https://static.igem.org/mediawiki/2010/0/0b/Freiburg10_pAAV_complete_mutations.png" | + | 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> | + | 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 | + | <p class="MsoCaption" style="text-indent: 0cm;"><a |
- | name="_Ref275820820"><span lang="EN-US">Figure </span></a><span | + | name="_Ref275820820"><span lang="EN-US">Figure </span></a><span |
- | lang="EN-US">1</span> <span | + | lang="EN-US">1</span> <span |
- | style="font-size: 8pt; color: windowtext; font-weight: normal;" | + | style="font-size: 8pt; color: windowtext; font-weight: normal;" |
- | lang="EN-US">Mutations implemented into <i>RepVP123</i> | + | lang="EN-US">Mutations implemented into <i>RepVP123</i> |
in order to establish both iGEM standard and loop insertion capability. | in order to establish both iGEM standard and loop insertion capability. | ||
Green arrows indicate integrated restriction sites, red red arrows | Green arrows indicate integrated restriction sites, red red arrows | ||
indicate deleted restriction sites. KpnI was deleted first and | indicate deleted restriction sites. KpnI was deleted first and | ||
reinstated afterwards. (see text).</span></p> | reinstated afterwards. (see text).</span></p> | ||
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | </tbody> | + | </tbody> |
</table> | </table> | ||
<span | <span | ||
- | style="font-size: 8pt; line-height: 150%; font-family: "Calibri","sans-serif";" | + | style="font-size: 8pt; line-height: 150%; font-family: "Calibri","sans-serif";" |
- | lang="EN-US"><br style="page-break-before: always;" clear="all"> | + | lang="EN-US"><br style="page-break-before: always;" |
+ | clear="all"> | ||
</span> | </span> | ||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
<p class="MsoNormal" | <p class="MsoNormal" | ||
- | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span | + | style="text-indent: 0cm; line-height: 150%;"><span |
- | style="font-size: 10pt; color: black;" lang="EN-US">Plasmid | + | style="font-size: 8pt; line-height: 150%;" lang="EN-US"> </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> | name:</span></p> | ||
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | width="103"> |
- | <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 |
- | style="font-size: 10pt; color: black;" lang="EN-US">Functionality | + | style="font-size: 10pt; color: black;" lang="EN-US">Functionality |
(determinded in cell culture via transduction and flow cytometry ):</span></p> | (determinded in cell culture via transduction and flow cytometry ):</span></p> | ||
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | width="104"> |
- | <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 |
- | style="font-size: 10pt; color: black;" lang="EN-US">4x | + | style="font-size: 10pt; color: black;" lang="EN-US">4x |
mutations (PstI (310), BamHI (859), SalI (1239), PstI (4073))</span></p> | mutations (PstI (310), BamHI (859), SalI (1239), PstI (4073))</span></p> | ||
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | width="57"> |
- | <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 |
- | style="font-size: 10pt; color: black;">inserted <i>rep</i> | + | style="font-size: 10pt; color: black;">inserted <i>rep</i> |
fragment</span></p> | fragment</span></p> | ||
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | width="66"> |
- | <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 |
- | style="font-size: 10pt; color: black;">inserted<i> cap</i> | + | style="font-size: 10pt; color: black;">inserted<i> cap</i> |
fragment</span></p> | fragment</span></p> | ||
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | width="67"> |
- | <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 |
- | style="font-size: 10pt; color: black;">reinstated KpnI</span></p> | + | style="font-size: 10pt; color: black;">reinstated KpnI</span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | width="32"> |
- | <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 |
- | style="font-size: 10pt; color: black;">pAAV</span></p> | + | style="font-size: 10pt; color: black;">pAAV</span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | width="70"> |
- | <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 |
- | style="font-size: 10pt; color: black;">pSB1C3_001</span></p> | + | style="font-size: 10pt; color: black;">pSB1C3_001</span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | width="37"> |
- | <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 |
- | style="font-size: 10pt; color: black;">HSPG-ko</span></p> | + | style="font-size: 10pt; color: black;">HSPG-ko</span></p> |
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | <tr style="height: 15pt;"> | + | <tr style="height: 15pt;"> |
- | <td | + | <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;" | + | 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"> | + | width="115"> |
- | <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 |
- | style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC | + | style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC |
(wild-type)</span></p> | (wild-type)</span></p> | ||
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | width="103"> |
- | <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 |
- | style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p> | + | style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | width="104"> |
- | <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 |
- | style="font-size: 10pt; color: black;" lang="EN-US"> </span></p> | + | style="font-size: 10pt; color: black;" lang="EN-US"> </span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="57"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;"> </span></p> | + | align="left"><span style="font-size: 10pt; color: black;"> </span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="66"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;"> </span></p> | + | align="left"><span style="font-size: 10pt; color: black;"> </span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="67"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;"> </span></p> | + | align="left"><span style="font-size: 10pt; color: black;"> </span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="32"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;">x</span></p> | + | align="left"><span style="font-size: 10pt; color: black;">x</span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="70"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;"> </span></p> | + | align="left"><span style="font-size: 10pt; color: black;"> </span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="37"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;"> </span></p> | + | align="left"><span style="font-size: 10pt; color: black;"> </span></p> |
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | <tr style="height: 15pt;"> | + | <tr style="height: 15pt;"> |
- | <td | + | <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;" | + | 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"> | + | width="115"> |
- | <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 |
- | style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC_4x | + | style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC_4x |
mutant</span></p> | mutant</span></p> | ||
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | width="103"> |
- | <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 |
- | style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p> | + | style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | width="104"> |
- | <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 |
- | style="font-size: 10pt; color: black;" lang="EN-US">x</span></p> | + | style="font-size: 10pt; color: black;" lang="EN-US">x</span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="57"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;"> </span></p> | + | align="left"><span style="font-size: 10pt; color: black;"> </span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="66"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;"> </span></p> | + | align="left"><span style="font-size: 10pt; color: black;"> </span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="67"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;"> </span></p> | + | align="left"><span style="font-size: 10pt; color: black;"> </span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="32"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;">x</span></p> | + | align="left"><span style="font-size: 10pt; color: black;">x</span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="70"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;"> </span></p> | + | align="left"><span style="font-size: 10pt; color: black;"> </span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="37"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;"> </span></p> | + | align="left"><span style="font-size: 10pt; color: black;"> </span></p> |
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | <tr style="height: 15pt;"> | + | <tr style="height: 15pt;"> |
- | <td | + | <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;" | + | 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"> | + | width="115"> |
- | <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 |
- | style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC_inserts </span></p> | + | style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC_inserts |
- | </td> | + | </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;" | + | <td |
- | width="103"> | + | 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;" |
- | <p class="MsoNormal" | + | width="103"> |
- | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span | + | <p class="MsoNormal" |
- | style="font-size: 10pt; color: black;" lang="EN-US">no</span></p> | + | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span |
- | </td> | + | 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;" | + | <td |
- | width="104"> | + | 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;" |
- | <p class="MsoNormal" | + | width="104"> |
- | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span | + | <p class="MsoNormal" |
- | style="font-size: 10pt; color: black;" lang="EN-US">x</span></p> | + | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span |
- | </td> | + | 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;" | + | <td |
- | nowrap="nowrap" valign="bottom" width="57"> | + | 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;" |
- | <p class="MsoNormal" | + | nowrap="nowrap" valign="bottom" width="57"> |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | <p class="MsoNormal" |
- | align="left"><span style="font-size: 10pt; color: black;">x</span></p> | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | </td> | + | 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;" | + | <td |
- | nowrap="nowrap" valign="bottom" width="66"> | + | 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;" |
- | <p class="MsoNormal" | + | nowrap="nowrap" valign="bottom" width="66"> |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | <p class="MsoNormal" |
- | align="left"><span style="font-size: 10pt; color: black;">x</span></p> | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | </td> | + | 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;" | + | <td |
- | nowrap="nowrap" valign="bottom" width="67"> | + | 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;" |
- | <p class="MsoNormal" | + | nowrap="nowrap" valign="bottom" width="67"> |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | <p class="MsoNormal" |
- | align="left"><span style="font-size: 10pt; color: black;"> </span></p> | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | </td> | + | align="left"><span style="font-size: 10pt; color: black;"> </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;" | + | <td |
- | nowrap="nowrap" valign="bottom" width="32"> | + | 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;" |
- | <p class="MsoNormal" | + | nowrap="nowrap" valign="bottom" width="32"> |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | <p class="MsoNormal" |
- | align="left"><span style="font-size: 10pt; color: black;">x</span></p> | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | </td> | + | 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;" | + | <td |
- | nowrap="nowrap" valign="bottom" width="70"> | + | 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;" |
- | <p class="MsoNormal" | + | nowrap="nowrap" valign="bottom" width="70"> |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | <p class="MsoNormal" |
- | align="left"><span style="font-size: 10pt; color: black;"> </span></p> | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | </td> | + | align="left"><span style="font-size: 10pt; color: black;"> </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;" | + | <td |
- | nowrap="nowrap" valign="bottom" width="37"> | + | 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;" |
- | <p class="MsoNormal" | + | nowrap="nowrap" valign="bottom" width="37"> |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | <p class="MsoNormal" |
- | align="left"><span style="font-size: 10pt; color: black;"> </span></p> | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | </td> | + | align="left"><span style="font-size: 10pt; color: black;"> </span></p> |
- | </tr> | + | </td> |
- | <tr style="height: 15pt;"> | + | </tr> |
- | <td | + | <tr style="height: 15pt;"> |
- | 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;" | + | <td |
- | width="115"> | + | 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;" |
- | <p class="MsoNormal" | + | width="115"> |
- | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span | + | <p class="MsoNormal" |
- | style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC_Cap</span></p> | + | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span |
- | </td> | + | 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;" | + | <td |
- | width="103"> | + | 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;" |
- | <p class="MsoNormal" | + | width="103"> |
- | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span | + | <p class="MsoNormal" |
- | style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p> | + | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span |
- | </td> | + | 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;" | + | <td |
- | width="104"> | + | 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;" |
- | <p class="MsoNormal" | + | width="104"> |
- | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span | + | <p class="MsoNormal" |
- | style="font-size: 10pt; color: black;" lang="EN-US">x</span></p> | + | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span |
- | </td> | + | 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;" | + | <td |
- | nowrap="nowrap" valign="bottom" width="57"> | + | 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;" |
- | <p class="MsoNormal" | + | nowrap="nowrap" valign="bottom" width="57"> |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | <p class="MsoNormal" |
- | align="left"><span style="font-size: 10pt; color: black;"> </span></p> | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | </td> | + | align="left"><span style="font-size: 10pt; color: black;"> </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;" | + | <td |
- | nowrap="nowrap" valign="bottom" width="66"> | + | 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;" |
- | <p class="MsoNormal" | + | nowrap="nowrap" valign="bottom" width="66"> |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | <p class="MsoNormal" |
- | align="left"><span style="font-size: 10pt; color: black;">x</span></p> | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | </td> | + | 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;" | + | <td |
- | nowrap="nowrap" valign="bottom" width="67"> | + | 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;" |
- | <p class="MsoNormal" | + | nowrap="nowrap" valign="bottom" width="67"> |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | <p class="MsoNormal" |
- | align="left"><span style="font-size: 10pt; color: black;"> </span></p> | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | </td> | + | align="left"><span style="font-size: 10pt; color: black;"> </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;" | + | <td |
- | nowrap="nowrap" valign="bottom" width="32"> | + | 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;" |
- | <p class="MsoNormal" | + | nowrap="nowrap" valign="bottom" width="32"> |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | <p class="MsoNormal" |
- | align="left"><span style="font-size: 10pt; color: black;">x</span></p> | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | </td> | + | 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;" | + | <td |
- | nowrap="nowrap" valign="bottom" width="70"> | + | 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;" |
- | <p class="MsoNormal" | + | nowrap="nowrap" valign="bottom" width="70"> |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | <p class="MsoNormal" |
- | align="left"><span style="font-size: 10pt; color: black;"> </span></p> | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | </td> | + | align="left"><span style="font-size: 10pt; color: black;"> </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;" | + | <td |
- | nowrap="nowrap" valign="bottom" width="37"> | + | 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;" |
- | <p class="MsoNormal" | + | nowrap="nowrap" valign="bottom" width="37"> |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | <p class="MsoNormal" |
- | align="left"><span style="font-size: 10pt; color: black;"> </span></p> | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | </td> | + | align="left"><span style="font-size: 10pt; color: black;"> </span></p> |
- | </tr> | + | </td> |
- | <tr style="height: 15pt;"> | + | </tr> |
- | <td | + | <tr style="height: 15pt;"> |
- | 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;" | + | <td |
- | width="115"> | + | 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;" |
- | <p class="MsoNormal" | + | width="115"> |
- | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span | + | <p class="MsoNormal" |
- | style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC_RepVP123</span></p> | + | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span |
- | </td> | + | 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;" | + | <td |
- | width="103"> | + | 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;" |
- | <p class="MsoNormal" | + | width="103"> |
- | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span | + | <p class="MsoNormal" |
- | style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p> | + | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span |
- | </td> | + | 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;" | + | <td |
- | width="104"> | + | 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;" |
- | <p class="MsoNormal" | + | width="104"> |
- | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span | + | <p class="MsoNormal" |
- | style="font-size: 10pt; color: black;" lang="EN-US">x</span></p> | + | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span |
- | </td> | + | 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;" | + | <td |
- | nowrap="nowrap" valign="bottom" width="57"> | + | 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;" |
- | <p class="MsoNormal" | + | nowrap="nowrap" valign="bottom" width="57"> |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | <p class="MsoNormal" |
- | align="left"><span style="font-size: 10pt; color: black;">x</span></p> | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | </td> | + | 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;" | + | <td |
- | nowrap="nowrap" valign="bottom" width="66"> | + | 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;" |
- | <p class="MsoNormal" | + | nowrap="nowrap" valign="bottom" width="66"> |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | <p class="MsoNormal" |
- | align="left"><span style="font-size: 10pt; color: black;">x</span></p> | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | </td> | + | 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;" | + | <td |
- | nowrap="nowrap" valign="bottom" width="67"> | + | 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;" |
- | <p class="MsoNormal" | + | nowrap="nowrap" valign="bottom" width="67"> |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | <p class="MsoNormal" |
- | align="left"><span style="font-size: 10pt; color: black;">x</span></p> | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | </td> | + | 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;" | + | <td |
- | nowrap="nowrap" valign="bottom" width="32"> | + | 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;" |
- | <p class="MsoNormal" | + | nowrap="nowrap" valign="bottom" width="32"> |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | <p class="MsoNormal" |
- | align="left"><span style="font-size: 10pt; color: black;">x</span></p> | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | </td> | + | 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;" | + | <td |
- | nowrap="nowrap" valign="bottom" width="70"> | + | 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;" |
- | <p class="MsoNormal" | + | nowrap="nowrap" valign="bottom" width="70"> |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | <p class="MsoNormal" |
- | align="left"><span style="font-size: 10pt; color: black;"> </span></p> | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | </td> | + | align="left"><span style="font-size: 10pt; color: black;"> </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;" | + | <td |
- | nowrap="nowrap" valign="bottom" width="37"> | + | 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;" |
- | <p class="MsoNormal" | + | nowrap="nowrap" valign="bottom" width="37"> |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | <p class="MsoNormal" |
- | align="left"><span style="font-size: 10pt; color: black;"> </span></p> | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | </td> | + | align="left"><span style="font-size: 10pt; color: black;"> </span></p> |
- | </tr> | + | </td> |
- | <tr style="height: 18pt;"> | + | </tr> |
- | <td | + | <tr style="height: 18pt;"> |
- | 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;" | + | <td |
- | width="115"> | + | 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;" |
- | <p class="MsoNormal" | + | width="115"> |
- | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span | + | <p class="MsoNormal" |
- | style="font-size: 10pt; color: black;" lang="EN-US">pSB1C3_RepVP123_ | + | 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> | p5TATAless</span></p> | ||
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | width="103"> |
- | <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 |
- | style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p> | + | style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | width="104"> |
- | <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 |
- | style="font-size: 10pt; color: black;" lang="EN-US">x</span></p> | + | style="font-size: 10pt; color: black;" lang="EN-US">x</span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="57"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;">x</span></p> | + | align="left"><span style="font-size: 10pt; color: black;">x</span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="66"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;">x</span></p> | + | align="left"><span style="font-size: 10pt; color: black;">x</span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="67"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;">x</span></p> | + | align="left"><span style="font-size: 10pt; color: black;">x</span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="32"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;"> </span></p> | + | align="left"><span style="font-size: 10pt; color: black;"> </span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="70"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;">x</span></p> | + | align="left"><span style="font-size: 10pt; color: black;">x</span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="37"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;"> </span></p> | + | align="left"><span style="font-size: 10pt; color: black;"> </span></p> |
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | <tr style="height: 27pt;"> | + | <tr style="height: 27pt;"> |
- | <td | + | <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;" | + | 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"> | + | width="115"> |
- | <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 |
- | style="font-size: 10pt; color: black;" lang="EN-US">pSB1C3_RepVP123_ | + | style="font-size: 10pt; color: black;" lang="EN-US">pSB1C3_RepVP123_ |
HSPG-ko_p5TATAless</span></p> | HSPG-ko_p5TATAless</span></p> | ||
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | width="103"> |
- | <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 |
- | style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p> | + | style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | width="104"> |
- | <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 |
- | style="font-size: 10pt; color: black;" lang="EN-US">x</span></p> | + | style="font-size: 10pt; color: black;" lang="EN-US">x</span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="57"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;">x</span></p> | + | align="left"><span style="font-size: 10pt; color: black;">x</span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="66"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;">x</span></p> | + | align="left"><span style="font-size: 10pt; color: black;">x</span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="67"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;">x</span></p> | + | align="left"><span style="font-size: 10pt; color: black;">x</span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="32"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;"> </span></p> | + | align="left"><span style="font-size: 10pt; color: black;"> </span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="70"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" | + | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;" |
- | align="left"><span style="font-size: 10pt; color: black;">x</span></p> | + | align="left"><span style="font-size: 10pt; color: black;">x</span></p> |
- | </td> | + | </td> |
- | <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;" | + | 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"> | + | nowrap="nowrap" valign="bottom" width="37"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal; page-break-after: avoid;" | + | 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> | + | align="left"><span style="font-size: 10pt; color: black;">x</span></p> |
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | </tbody> | + | </tbody> |
</table> | </table> | ||
- | <p class="MsoCaption" style="text-align: left;" align="left"><span | + | <p class="MsoCaption" style="text-align: left;" |
- | lang="EN-US">Figure </span><span lang="EN-US">2</span> <span | + | align="left"><span lang="EN-US">Figure </span><span |
- | style="color: windowtext; font-weight: normal;" lang="EN-US">Table | + | 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> | contains complete overview about all plasmids containing <i>RepVP123</i> | ||
which | which | ||
were used by iGEM team Freiburg_Bioware 2010.</span></p> | were used by iGEM team Freiburg_Bioware 2010.</span></p> | ||
- | <h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275885923"></a><a | + | <h3 style="margin-left: 0cm; text-indent: 0cm;"><a |
- | name="_Toc275817882"><span lang="EN-US">Modularization: Removing iGEM | + | name="_Toc275885923"></a><a name="_Toc275817882"><span |
+ | lang="EN-US">Modularization: Removing iGEM | ||
restriction | restriction | ||
sites and establishing loop insertion capability</span></a></h3> | sites and establishing loop insertion capability</span></a></h3> | ||
- | <h4 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275885924"></a><a | + | <h4 style="margin-left: 0cm; text-indent: 0cm;"><a |
- | name="_Toc275817883"><span lang="EN-US">Modifications in Rep</span></a></h4> | + | name="_Toc275885924"></a><a name="_Toc275817883"><span |
+ | lang="EN-US">Modifications in Rep</span></a></h4> | ||
<table class="MsoTableGrid" | <table class="MsoTableGrid" | ||
- | style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;" | + | style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;" |
- | border="0" 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; vertical-align: top; width: 460.6pt;"> | + | style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 460.6pt;"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="text-align: center; text-indent: 0cm; page-break-after: avoid;" | + | style="text-align: center; text-indent: 0cm; page-break-after: avoid;" |
- | align="center"><img style="width: 576px; height: 191px;" id="Picture 4" | + | align="center"><img style="width: 576px; height: 191px;" |
- | src="https://static.igem.org/mediawiki/2010/b/bc/Freiburg10_rep_synthetic_gene_fragment.png" | + | id="Picture 4" |
- | alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\Rep_synthesis_marked.jpg"></p> | + | src="https://static.igem.org/mediawiki/2010/b/bc/Freiburg10_rep_synthetic_gene_fragment.png" |
- | <p class="MsoCaption"><a name="_Ref275818017"></a><a | + | alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\Rep_synthesis_marked.jpg"></p> |
- | name="_Ref275818042"><span lang="EN-US">Figure </span></a><span | + | <p class="MsoCaption"><a name="_Ref275818017"></a><a |
- | lang="EN-US">3</span> <span | + | name="_Ref275818042"><span lang="EN-US">Figure </span></a><span |
- | style="font-size: 8pt; color: windowtext; font-weight: normal;" | + | lang="EN-US">3</span> <span |
- | lang="EN-US">Restriction sites within the wild-type <i>rep</i> | + | style="font-size: 8pt; color: windowtext; font-weight: normal;" |
- | gene sequence, which were removed via cloning of synthetized <i>rep </i>gene | + | 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 | fragment into the plasmid. The red box indicates | ||
the region spanned by the synthetic sequence.</span></p> | the region spanned by the synthetic sequence.</span></p> | ||
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | </tbody> | + | </tbody> |
</table> | </table> | ||
- | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">Making | + | <p class="MsoNormal" style="text-indent: 0cm;"><span |
+ | lang="EN-US">Making | ||
the <i>RepVP123</i> | the <i>RepVP123</i> | ||
wild-type compatible with the iGEM standards required the removal of | wild-type compatible with the iGEM standards required the removal of | ||
Line 1,709: | Line 1,883: | ||
each other plus | each other plus | ||
an additional KpnI restriction site which was also not desired (see </span><span | an additional KpnI restriction site which was also not desired (see </span><span | ||
- | lang="EN-US">Figure 2</span><span lang="EN-US">). | + | lang="EN-US">Figure 2</span><span lang="EN-US">). |
This gene fragment was cloned into | This gene fragment was cloned into | ||
the <i>rep</i> gene using HindIII and SwaI, which are | the <i>rep</i> gene using HindIII and SwaI, which are | ||
Line 1,718: | Line 1,892: | ||
genetically | genetically | ||
inserting the loop modifications in VP123.</span></p> | inserting the loop modifications in VP123.</span></p> | ||
- | <h4 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275885925"></a><a | + | <h4 style="margin-left: 0cm; text-indent: 0cm;"><a |
- | name="_Toc275817884"><span lang="EN-US">Modifications in VP123</span></a></h4> | + | name="_Toc275885925"></a><a name="_Toc275817884"><span |
- | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">In | + | lang="EN-US">Modifications in VP123</span></a></h4> |
+ | <p class="MsoNormal" style="text-indent: 0cm;"><span | ||
+ | lang="EN-US">In | ||
order to | order to | ||
implement the restriction sites necessary for targeting via loop | implement the restriction sites necessary for targeting via loop | ||
Line 1,732: | Line 1,908: | ||
as well.</span></p> | as well.</span></p> | ||
<table class="MsoTableGrid" | <table class="MsoTableGrid" | ||
- | style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;" | + | style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;" |
- | border="0" 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; vertical-align: top; width: 460.6pt;"> | + | style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 460.6pt;"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="text-align: center; text-indent: 0cm; page-break-after: avoid;" | + | style="text-align: center; text-indent: 0cm; page-break-after: avoid;" |
- | align="center"><img style="width: 574px; height: 218px;" id="Picture 5" | + | align="center"><img style="width: 574px; height: 218px;" |
- | src="https://static.igem.org/mediawiki/2010/c/cd/Freiburg10_cap_synthetic_gene_fragment.png" | + | id="Picture 5" |
- | alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\Cap_synthesis_marked.jpg"></p> | + | src="https://static.igem.org/mediawiki/2010/c/cd/Freiburg10_cap_synthetic_gene_fragment.png" |
- | <p class="MsoCaption"><span lang="EN-US">Figure </span><span | + | alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\Cap_synthesis_marked.jpg"></p> |
- | lang="EN-US">4</span> <span | + | <p class="MsoCaption"><span lang="EN-US">Figure |
- | style="font-size: 8pt; color: windowtext; font-weight: normal;" | + | </span><span lang="EN-US">4</span> <span |
- | lang="EN-US">Restriction sites within <i>cap</i> | + | 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> | sequence showing introduced loop insertion restriction sites into <i>cap</i> | ||
to enable cloning of targeting or purification motifs into both 453 and | to enable cloning of targeting or purification motifs into both 453 and | ||
587 loops. Again, the red box indicates gene sequence which was | 587 loops. Again, the red box indicates gene sequence which was | ||
synthetized.</span></p> | synthetized.</span></p> | ||
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | </tbody> | + | </tbody> |
</table> | </table> | ||
- | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US"> </span></p> | + | <p class="MsoNormal" style="text-indent: 0cm;"><span |
+ | lang="EN-US"> </span></p> | ||
<table class="MsoTableGrid" | <table class="MsoTableGrid" | ||
- | style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;" | + | style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;" |
- | border="0" cellpadding="0" cellspacing="0"> | + | border="0" cellpadding="0" cellspacing="0"> |
- | <tbody> | + | <tbody> |
- | <tr> | + | <tr> |
- | <td | + | <td |
- | style="border: 1pt solid windowtext; padding: 0cm 3.5pt; vertical-align: top; width: 460.6pt;"> | + | style="border: 1pt solid windowtext; padding: 0cm 3.5pt; vertical-align: top; width: 460.6pt;"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="text-align: center; text-indent: 0cm; page-break-after: avoid;" | + | style="text-align: center; text-indent: 0cm; page-break-after: avoid;" |
- | align="center"><img style="width: 511px; height: 302px;" alt="" | + | align="center"><img style="width: 511px; height: 302px;" |
- | id="Chart 7" | + | alt="" id="Chart 7" |
- | src="https://static.igem.org/mediawiki/2010/6/6b/Freiburg10_wt_vs_4xmutant.png"></p> | + | 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 | + | <p class="MsoCaption"><span lang="EN-US">Figure |
- | lang="EN-US">5</span> <span | + | </span><span lang="EN-US">5</span> <span |
- | style="color: windowtext; font-weight: normal;" lang="EN-US">Results | + | style="color: windowtext; font-weight: normal;" lang="EN-US">Results |
- | for transduction efficiency measured by flow cytometry. The tested <i>RepVP123</i> | + | 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 | containing four point mutation to delete iGEM and loop insertion | ||
restriction sites does not show any difference within mVenus expression | restriction sites does not show any difference within mVenus expression | ||
compared to the wild-type and therefore can be verified working.</span></p> | compared to the wild-type and therefore can be verified working.</span></p> | ||
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | </tbody> | + | </tbody> |
</table> | </table> | ||
- | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">Alongside | + | <p class="MsoNormal" style="text-indent: 0cm;"><span |
+ | lang="EN-US">Alongside | ||
to | to | ||
creating an iGEM compatible plasmid, infectivity of the modified | creating an iGEM compatible plasmid, infectivity of the modified | ||
Line 1,801: | Line 1,981: | ||
production (see extra topic “Rep proteins”).</span></p> | production (see extra topic “Rep proteins”).</span></p> | ||
<span | <span | ||
- | style="font-size: 11pt; line-height: 200%; font-family: "Calibri","sans-serif";" | + | style="font-size: 11pt; line-height: 200%; font-family: "Calibri","sans-serif";" |
- | lang="EN-US"><br style="page-break-before: always;" clear="all"> | + | lang="EN-US"><br style="page-break-before: always;" |
+ | clear="all"> | ||
</span> | </span> | ||
- | <p class="MsoNormal" style="text-indent: 0cm; text-align: center;"><span | + | <p class="MsoNormal" |
- | lang="EN-US"> <img alt="xxx" | + | style="text-indent: 0cm; text-align: center;"><span |
- | src="https://static.igem.org/mediawiki/2010/f/fd/Freiburg10_Freiburg10_facs_table1.png"><br> | + | lang="EN-US"> <img alt="xxx" |
+ | src="https://static.igem.org/mediawiki/2010/f/fd/Freiburg10_Freiburg10_facs_table1.png"><br> | ||
</span></p> | </span></p> | ||
<br> | <br> | ||
<p style="text-align: center;" class="MsoNormal"> <img | <p style="text-align: center;" class="MsoNormal"> <img | ||
- | style="width: 636px; height: 396px;" alt="" | + | style="width: 636px; height: 396px;" alt="" |
- | src="https://static.igem.org/mediawiki/2010/3/3a/Freiburg10_Freiburg10_facs_table2.png"></p> | + | src="https://static.igem.org/mediawiki/2010/3/3a/Freiburg10_Freiburg10_facs_table2.png"></p> |
<br> | <br> | ||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
<p class="MsoNormal" | <p class="MsoNormal" | ||
- | style="margin-bottom: 10pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;" | + | style="text-indent: 0cm; line-height: 150%;"><span |
- | align="center"><span style="font-size: 10pt;"><img | + | lang="EN-US"></span></p> |
- | style="width: 395px; height: 215px;" id="Chart 18" | + | <p class="MsoNormal" |
- | src="https://static.igem.org/mediawiki/2010/7/73/Freiburg10_KpnI_deleted.png" | + | style="text-indent: 0cm; line-height: 150%;"><span |
- | 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> | + | lang="EN-US"></span></p> |
- | <p style="text-align: justify;" class="MsoCaption"><span | + | <table |
- | lang="EN-US">Figure </span><span lang="EN-US">8</span> <span | + | style="width: 474px; text-align: left; margin-left: auto; margin-right: auto;" |
- | style="color: windowtext; font-weight: normal;" lang="EN-US">AAV-293 | + | 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, | cells were transfected with three plasmids pHelper, | ||
pSB1C3_[AAV2]-left-ITR_pCMV_betaglobin_mVenus _hGH_[AAV2]-right-ITR and | pSB1C3_[AAV2]-left-ITR_pCMV_betaglobin_mVenus _hGH_[AAV2]-right-ITR and | ||
Line 1,839: | Line 2,024: | ||
centrifugation followed by HT1080 transduction. mVenus expression of | centrifugation followed by HT1080 transduction. mVenus expression of | ||
viral genomes was determined by flow cytomery 24 hours post | viral genomes was determined by flow cytomery 24 hours post | ||
- | infection. Results show that insertion of both <i>rep</i> | + | 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> | and <i>cap</i> syntheses disrupts viral infectivity.</span></p> | ||
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | </tbody> | + | </tbody> |
</table> | </table> | ||
- | <p class="MsoNormal" style="text-indent: 0cm; line-height: 150%;"><span | + | <p class="MsoNormal" |
- | lang="EN-US"></span></p> | + | style="text-indent: 0cm; line-height: 150%;"><span |
- | <p class="MsoNormal" style="text-indent: 0cm; line-height: 150%;"><span | + | lang="EN-US"></span></p> |
- | lang="EN-US">Therefore, | + | <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> | additional constructs were designed containing only the synthetic <i>cap</i> | ||
sequence or both, the synthesized sequences plus a re-mutation of KpnI | sequence or both, the synthesized sequences plus a re-mutation of KpnI | ||
Line 1,861: | Line 2,052: | ||
<div align="center"> | <div align="center"> | ||
<table class="MsoTableGrid" | <table class="MsoTableGrid" | ||
- | style="border: medium none ; border-collapse: collapse;" border="0" | + | style="border: medium none ; border-collapse: collapse;" |
- | cellpadding="0" cellspacing="0"> | + | border="0" cellpadding="0" cellspacing="0"> |
- | <tbody> | + | <tbody> |
- | <tr style="height: 177.6pt;"> | + | <tr style="height: 177.6pt;"> |
- | <td | + | <td |
- | style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 464.4pt; height: 177.6pt;"> | + | style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 464.4pt; height: 177.6pt;"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="margin-bottom: 10pt; text-align: center; text-indent: 0cm; line-height: 115%; page-break-after: avoid;" | + | 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="" | + | align="center"><img style="width: 625px; height: 233px;" |
- | id="Chart 3" | + | alt="" id="Chart 3" |
- | src="https://static.igem.org/mediawiki/2010/6/62/Freiburg10_KpnI_reinstated.png"></p> | + | src="https://static.igem.org/mediawiki/2010/6/62/Freiburg10_KpnI_reinstated.png"></p> |
- | <p class="MsoCaption"><span lang="EN-US">Figure </span><span | + | <p class="MsoCaption"><span lang="EN-US">Figure |
- | lang="EN-US">9</span> <span lang="EN-US">F</span><span | + | </span><span lang="EN-US">9</span> <span |
- | style="color: windowtext; font-weight: normal;" lang="EN-US">luorescence | + | 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 | of cells transduced with mVenus carrying rAAV measured by flow | ||
cytometry. AAV-293 cells were transfected with three plasmids pHelper, | cytometry. AAV-293 cells were transfected with three plasmids pHelper, | ||
Line 1,883: | Line 2,075: | ||
transduction. mVenus expression of viral genomes was determined by flow | transduction. mVenus expression of viral genomes was determined by flow | ||
cytometry 24 hours post infection. Results show that <i>cap </i>integration | cytometry 24 hours post infection. Results show that <i>cap </i>integration | ||
- | does not influence infectivity. Recreation | + | 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 | of KpnI within <i>rep</i> splice site recovers | ||
transduction efficiency.</span></p> | transduction efficiency.</span></p> | ||
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | </tbody> | + | </tbody> |
</table> | </table> | ||
</div> | </div> | ||
- | <p class="MsoNormal" style="text-indent: 0cm; line-height: 150%;"><span | + | <p class="MsoNormal" |
- | lang="EN-US"> </span></p> | + | style="text-indent: 0cm; line-height: 150%;"><span |
- | <h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275885926"></a><a | + | lang="EN-US"> </span></p> |
- | name="_Toc275817885"><span lang="EN-US">Modularization: Adapting | + | <h3 style="margin-left: 0cm; text-indent: 0cm;"><a |
+ | name="_Toc275885926"></a><a name="_Toc275817885"><span | ||
+ | lang="EN-US">Modularization: Adapting | ||
pSB1C3 to loop | pSB1C3 to loop | ||
insertions – pSB1C3_001</span></a></h3> | insertions – pSB1C3_001</span></a></h3> | ||
- | <p class="MsoNormal" style="text-indent: 0cm; line-height: 150%;"><span | + | <p class="MsoNormal" |
- | lang="EN-US">To | + | style="text-indent: 0cm; line-height: 150%;"><span |
+ | lang="EN-US">To | ||
fulfill iGEM requirements all plasmids need to be submitted in pSB1C3, | fulfill iGEM requirements all plasmids need to be submitted in pSB1C3, | ||
therefore primers were ordered for amplifying <i>RepVP123</i> | therefore primers were ordered for amplifying <i>RepVP123</i> | ||
Line 1,916: | Line 2,115: | ||
make SspI and PvuII single-cutters (see method development).</span></p> | make SspI and PvuII single-cutters (see method development).</span></p> | ||
<table class="MsoTableGrid" | <table class="MsoTableGrid" | ||
- | style="border: medium none ; margin-left: auto; border-collapse: collapse; text-align: left; margin-right: auto;" | + | style="border: medium none ; margin-left: auto; border-collapse: collapse; text-align: left; margin-right: auto;" |
- | border="0" 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; vertical-align: top; width: 460.6pt;"> | + | style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 460.6pt;"> |
- | <p class="MsoNormal" | + | <p class="MsoNormal" |
- | style="text-align: center; text-indent: 0cm; page-break-after: avoid;" | + | style="text-align: center; text-indent: 0cm; page-break-after: avoid;" |
- | align="center"><img style="width: 584px; height: 278px;" | + | align="center"><img style="width: 584px; height: 278px;" |
- | id="Picture 16" | + | id="Picture 16" |
- | src="https://static.igem.org/mediawiki/2010/d/d1/Freiburg10_pSB1C3_001_mutations.png" | + | src="https://static.igem.org/mediawiki/2010/d/d1/Freiburg10_pSB1C3_001_mutations.png" |
- | alt=""></p> | + | alt=""></p> |
- | <p class="MsoCaption"><span lang="EN-US">Figure </span><span | + | <p class="MsoCaption"><span lang="EN-US">Figure |
- | lang="EN-US">10</span> <span | + | </span><span lang="EN-US">10</span> <span |
- | style="color: windowtext; font-weight: normal;" lang="EN-US">Comparison | + | style="color: windowtext; font-weight: normal;" lang="EN-US">Comparison |
of pSB1C3 (upper row) and pSB1C3_001 (lower row). Deletions of SspI and | of pSB1C3 (upper row) and pSB1C3_001 (lower row). Deletions of SspI and | ||
PvuII are marked by red boxes.</span></p> | PvuII are marked by red boxes.</span></p> | ||
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | </tbody> | + | </tbody> |
</table> | </table> | ||
<p class="MsoNormal"><i><span lang="EN-US">RepVP123</span></i><span | <p class="MsoNormal"><i><span lang="EN-US">RepVP123</span></i><span | ||
- | lang="EN-US"> | + | lang="EN-US"> |
containing both <i>rep</i> and <i>cap</i> | containing both <i>rep</i> and <i>cap</i> | ||
synthetic gene fragments including | synthetic gene fragments including | ||
Line 1,956: | Line 2,155: | ||
approximately 1000 base pairs.</span></p> | approximately 1000 base pairs.</span></p> | ||
<span | <span | ||
- | style="font-size: 11pt; line-height: 200%; font-family: "Calibri","sans-serif";" | + | style="font-size: 11pt; line-height: 200%; font-family: "Calibri","sans-serif";" |
- | lang="EN-US"></span><span lang="EN-US"> </span> | + | lang="EN-US"></span><span lang="EN-US"> </span> |
<table | <table | ||
- | style="border: medium none ; width: 466px; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;" | + | 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"> | + | class="MsoTableGrid" border="0" cellpadding="0" |
- | <tbody> | + | cellspacing="0"> |
- | <tr style="height: 209.85pt;"> | + | <tbody> |
- | <td | + | <tr style="height: 209.85pt;"> |
- | style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 4661px; height: 209.85pt;"> | + | <td |
- | <p class="MsoNormal" | + | style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 4661px; height: 209.85pt;"> |
- | style="text-align: center; text-indent: 0cm; page-break-after: avoid;" | + | <p class="MsoNormal" |
- | align="center"><span style="font-size: 10pt; line-height: 200%;"><img | + | style="text-align: center; text-indent: 0cm; page-break-after: avoid;" |
- | style="width: 516px; height: 258px;" alt="" id="Chart 15" | + | align="center"><span |
- | src="https://static.igem.org/mediawiki/2010/2/2b/Freiburg10_pAAV_pSB1C3_001.png"></span></p> | + | style="font-size: 10pt; line-height: 200%;"><img |
- | <p class="MsoCaption"><span lang="EN-US">Figure </span><span | + | style="width: 516px; height: 258px;" alt="" id="Chart 15" |
- | lang="EN-US">11</span> <span | + | src="https://static.igem.org/mediawiki/2010/2/2b/Freiburg10_pAAV_pSB1C3_001.png"></span></p> |
- | style="color: windowtext; font-weight: normal;" lang="EN-US">AAV-293 | + | <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, | cells were transfected with three plasmids pHelper, | ||
pSB1C3_001_[AAV2]-Rep-VP123_p5-TATAless or pAAV_RC_IRCK and | pSB1C3_001_[AAV2]-Rep-VP123_p5-TATAless or pAAV_RC_IRCK and | ||
Line 1,980: | Line 2,181: | ||
freeze-thaw lysis and centrifugation followed by HT1080 transduction. | freeze-thaw lysis and centrifugation followed by HT1080 transduction. | ||
mVenus expression of viral genomes was determined by flow cytomery | mVenus expression of viral genomes was determined by flow cytomery | ||
- | after 24 hours post infection. Results showed functionality of <i>RepVP123</i> | + | 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"> Results | ||
+ | showed functionality of <i>RepVP123</i> | ||
within pSB1C3_001 vector and additionally increased transduction | within pSB1C3_001 vector and additionally increased transduction | ||
efficiency.</span></p> | efficiency.</span></p> | ||
- | </td> | + | </td> |
- | </tr> | + | </tr> |
- | </tbody> | + | </tbody> |
</table> | </table> | ||
- | <h4 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275885927"></a><a | + | <h4 style="margin-left: 0cm; text-indent: 0cm;"><a |
- | name="_Toc275817886"><span lang="EN-US">Turning-off natural tropism: | + | name="_Toc275885927"></a><a name="_Toc275817886"><span |
+ | lang="EN-US">Turning-off natural tropism: | ||
HSPG-knock-out</span></a></h4> | HSPG-knock-out</span></a></h4> | ||
- | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">Shutting-down | + | <p class="MsoNormal" style="text-indent: 0cm;"><span |
+ | lang="EN-US">Shutting-down | ||
the | the | ||
natural viral tropism is essential for targeting specifically tumor | natural viral tropism is essential for targeting specifically tumor | ||
Line 2,014: | Line 2,221: | ||
rate | rate | ||
compared to unspecific infection of other cell types within an organism | 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> | + | (see </span><span lang="EN-US">Figure 12</span><span |
- | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">To | + | lang="EN-US">).</span></p> |
+ | <p class="MsoNormal" style="text-indent: 0cm;"><span | ||
+ | lang="EN-US">To | ||
quantify | quantify | ||
differences in infectivity, the infectious titer of viral particles | differences in infectivity, the infectious titer of viral particles | ||
Line 2,021: | Line 2,230: | ||
<i>RepVP123</i> with and without HSPG binding motif was | <i>RepVP123</i> with and without HSPG binding motif was | ||
determined by qPCR (see | determined by qPCR (see | ||
- | </span><span lang="EN-US">Figure 14</span><span lang="EN-US">) for | + | </span><span lang="EN-US">Figure 14</span><span |
+ | lang="EN-US">) for | ||
different cell | different cell | ||
lines. Results show that the implemented HSPG-knock-out verifies | lines. Results show that the implemented HSPG-knock-out verifies | ||
Line 2,028: | Line 2,238: | ||
with intact HSPG binding motif.</span></p> | with intact HSPG binding motif.</span></p> | ||
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- | style="width: 595px; height: 379px;" id="Picture 2" | + | style="width: 595px; height: 379px;" id="Picture 2" |
- | src="https://static.igem.org/mediawiki/2010/9/9d/Freiburg10_HSPG-ko_sequence.png" | + | 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> | + | 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 | + | <p class="MsoCaption" style="text-align: left;" |
- | lang="EN-US">Figure </span><span lang="EN-US">12</span> <span | + | align="left"><span lang="EN-US">Figure </span><span |
- | style="color: windowtext; font-weight: normal;" lang="EN-US">Alignment | + | 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 | of 587 loop within viral VP123: The upper sequence shows a strand | ||
containing the HSPG binding motif (AGA, in red boxes), the lower | containing the HSPG binding motif (AGA, in red boxes), the lower | ||
sequence contains the HSPG-ko introduced by the iGEM team | sequence contains the HSPG-ko introduced by the iGEM team | ||
Freiburg_Bioware 2010 (GCT and GCC, blue boxes).</span></p> | Freiburg_Bioware 2010 (GCT and GCC, blue boxes).</span></p> | ||
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- | align="center"><img style="width: 467px; height: 229px;" alt="" | + | align="center"><img style="width: 467px; height: 229px;" |
- | id="Chart 9" | + | alt="" id="Chart 9" |
- | src="https://static.igem.org/mediawiki/2010/0/01/Freiburg10_columns_HSPG_HSPG_ko.png"></p> | + | 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 | + | <p class="MsoCaption"><a name="_Ref275882130"><span |
- | </span></a><span lang="EN-US">13</span> <span | + | lang="EN-US">Figure |
- | style="color: windowtext; font-weight: normal;" lang="EN-US">Transduction | + | </span></a><span lang="EN-US">13</span> |
- | efficiency of HT1080 cells measured by flow cytometry. Knock-out of | + | <span style="color: windowtext; font-weight: normal;" |
+ | lang="EN-US">Transduction | ||
+ | efficiency of HT1080 cells measured by flow cytometry. </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> | HSPG binding motif greatly reduces transduction efficiency compared to <i>RepVP123</i> | ||
containing the motiv.</span></p> | containing the motiv.</span></p> | ||
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- | src="https://static.igem.org/mediawiki/2010/b/bc/Freiburg10_qPCR_HSPG_HSPG_ko.png"></p> | + | alt="" id="Chart 1" |
- | <p class="MsoCaption" style="text-align: center;" align="center"><a | + | src="https://static.igem.org/mediawiki/2010/b/bc/Freiburg10_qPCR_HSPG_HSPG_ko.png"></p> |
- | name="_Ref275883877"><span lang="EN-US">Figure </span></a><span | + | <p class="MsoCaption" style="text-align: center;" |
- | lang="EN-US">14</span> <span | + | align="center"><a name="_Ref275883877"><span |
- | style="color: windowtext; font-weight: normal;" lang="EN-US">Infectious | + | lang="EN-US">Figure </span></a><span |
+ | lang="EN-US">14</span> <span | ||
+ | style="color: windowtext; font-weight: normal;" lang="EN-US">Infectious | ||
titers of <i>RepVP123</i> with and without natural HSPG | titers of <i>RepVP123</i> with and without natural HSPG | ||
binding motif tested in different cell lines via qPCR. Shutting-down | binding motif tested in different cell lines via qPCR. Shutting-down | ||
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qPCR, which is probably related to poor transduction efficiency of A431 | qPCR, which is probably related to poor transduction efficiency of A431 | ||
cells.</span></p> | cells.</span></p> | ||
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Revision as of 01:45, 27 October 2010
Modularization
Contents
Modularization of the vectorplasmid
Introduction to Modularization of Vectorplasmid
Recombinant and Modular Vectorplasmid Carrying GOI
Cloning and Combination Strategies for the Vectorplasmid
Testing functionality of Assembled Vectorplasmid
Fluorescence Microscopy of Target Cells Demonstrates GOI Expression
Analysis of Target Cells by Flow Cytometry demonstrates GOI Expression
Influence of hGH terminator BioBrick on GOI Expression
Influence of Beta-globin intron Biobrick on GOI Expression
Functionality of the Full Assembled Vectorplasmid Demonstrated by GOI Expression
Modularization of the RepVP123 plasmids
Modularization: Removing iGEM restriction sites and establishing loop insertion capability
Modularization: Adapting pSB1C3 to loop insertions – pSB1C3_001
Turning-off natural tropism: HSPG-knock-out
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_K404101), a promoter (pCMV :BBa_K404102 or phTERT: BBa_K404106)) , the beta-globin intron (BBa_K404107), the gene of interests (fluorescent proteins mVenus: BBa_I757008 and mCherry: BBa_J06504, suicide genes mGMK_TK30: BBa_K404112, mGMK_SR39: BBa_K404315 and CD: BBa_K404112) and the hGH terminator (BBa_K404108).
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.
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.
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).
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
After assembly of plasmids containing all required elements (see Figure 1), functionality was tested in cell culture. AAV-293 cells stably expressing E1A and E1B proteins were transfected with three plasmids (pHelper, pRC, pGOI). Virus particles were harvested 72 hours post-transfection and the tumor cell line HT1080 was transduced with the recombinant viral vectors encapsidating the gene of interest mVenus (BBa_I757008).
The iGEM team Freiburg_Bioware 2010 compared the standard-plasmid containing a subcloned mVenus (pAAV_mVenus, derived from the Stratagene system) with the assembled plasmid pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (pSB1C3_mVenus: BBa_K404119). Fluorescence expression data obtained by flow cytometry analysis are shown in Figure 11 and Figure 12. Comparing mVenus expression of the standard plasmid and the modified, assembled plasmid reveals that biological functionality of the reassembled plasmid was confirmed.
pSB1C3_mVenus (BBa_K404119) |
pAAV_mVenus (Stratagene) |
Figure 12: Flow cytometry analysis of reassembled vectorplasmid (BBa_K404119) compared to standard plasmid provided by Stratagene. A: Gating non transduced cells (control); subcellular debris and clumps can be distinguished from single cells by size, estimated forward scatter (FS Lin) and granularity, estimated side scatter (SS Lin) B: Non transduced cells plotted against mVenus (Analytical gate was set such that 1% or fewer of negative control cells fell within the positive region (R5).C: Gating transduced cells (R2 ≙R14) (used plasmids for transfection: pGOI: pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (pSB1C3_mVenus: BBa_K404119), pHelper, pRC. D: Transduced cells plotted against mVenus, R10 comprised transduced cells, by detecting mVenus expression. E: Overlay of non-transduced (red) and transduced (green). F: Gating non transduced cells (control). G: Non-transduced cells plotted against mVenus (R5). H: Gating transduced cells (R14 ≙R2) (used plasmids for transfection: pGOI: pAAV_mVenus, pHelper). I: Transduced cells plotted against mVenus, R10 comprised transduced cells, by detecting mVenus expression. J: Overlay of non-transduced (red) and transduced (green) cells plotted against mVenus fluorescence. |
Figure 13: Flow cytometry analysis of reassembled vectorplasmid (BBa_K404119) compared to standard plasmid provided by Stratagene. Fluorescence of the standard plasmid pAAV_mVenus (Stratagene) and the recombinant pSB1C3_mVenus (BBa_K404119) construct was measured. As demonstrated mVenus expression is enhanced in the assembled plasmid (pSB1C3_mVenus) compared to the standard pAAV_mVenus construct. |
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 – Killing the tumor’.
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.
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.
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
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.
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”).
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.
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. |