Team:Freiburg Bioware/NoteBook/Labjournal

From 2010.igem.org

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<div class="box_labjournal_195">
<div class="box_labjournal_195">
<a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/March">
<a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/March">
<h2 style="margin-left: 5px;">March</h2>
<h2 style="margin-left: 5px;">March</h2>
<p class="boxes_labjournal_day">Labday 1</p>
<p class="boxes_labjournal_day">Labday 1</p>
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<img src="http://2010.igem.org/wiki/images/3/35/Freiburg_10_BioBrick_icon.png" class="image_labjournal" />
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<img src="http://2010.igem.org/wiki/images/b/b4/Freiburg_10_labjournal_march_v1.JPG" class="image_labjournal" title="Our project starts with first sequence analysis."/>
<p class="boxes_labjournal_text">...watching gene sequences...</p>
<p class="boxes_labjournal_text">...watching gene sequences...</p>
</a>
</a>
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<h2 style="margin-left: 5px;">April</h2>
<h2 style="margin-left: 5px;">April</h2>
<p class="boxes_labjournal_day">Labday 2- 5</p>
<p class="boxes_labjournal_day">Labday 2- 5</p>
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<img src="http://2010.igem.org/wiki/images/3/35/Freiburg_10_BioBrick_icon.png" class="image_labjournal" />
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<img src="http://2010.igem.org/wiki/images/f/fc/Freiburg_10_labjournal_april_v1.JPG" class="image_labjournal" title="Scanning for iGEM restriction sites:
 +
Vector plasmid; Thymidine kinase ;Cytosindeaminase" />
<p class="boxes_labjournal_text">...ordering AAV-2 Helper-free System...</p>
<p class="boxes_labjournal_text">...ordering AAV-2 Helper-free System...</p>
</a>
</a>
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<h2 style="margin-left: 5px;">May</h2>
<h2 style="margin-left: 5px;">May</h2>
<p class="boxes_labjournal_day">Labday 6- 17</p>
<p class="boxes_labjournal_day">Labday 6- 17</p>
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<img src="http://2010.igem.org/wiki/images/3/35/Freiburg_10_BioBrick_icon.png" class="image_labjournal" />
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<img src="http://2010.igem.org/wiki/images/0/0f/Freiburg_10_labjournal_may_v1.JPG" class="image_labjournal" title="We took a closer look to the theoretical DNA- and amino acid sequence of the ITRs, questioned the role of the β-globin intron, had a closer look to the CMV promoter and performed our first cloning attempt." />
<p class="boxes_labjournal_text">...theoretical cloning...</p>
<p class="boxes_labjournal_text">...theoretical cloning...</p>
</a>
</a>
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<h2 style="margin-left: 5px;">June</h2>
<h2 style="margin-left: 5px;">June</h2>
<p class="boxes_labjournal_day">Labday 18- 45</p>
<p class="boxes_labjournal_day">Labday 18- 45</p>
-
<img src="http://2010.igem.org/wiki/images/3/35/Freiburg_10_BioBrick_icon.png" class="image_labjournal" />
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<img src="http://2010.igem.org/wiki/images/3/37/Freiburg_10_labjournal_june_v1.jpg" class="image_labjournal" title="
 +
We conducted our first site-directed mutagenesis. First cell culture steps: Splitting and seeding of AAV 293 and HT1080 cells; Calcium phosphate transfection; Transduction with virus particles containing YFP encoding sequence; Microscopy: Fluorescent transduced HT1080 cells. We planned to determine infectious virus titer via quantitative real-time PCR. For this purpose primers were designed, transduced HT1080 cells were harvested and prepared. Theoretical studies of the AAV structure: Impressions" />
<p class="boxes_labjournal_text">...first transduced cells...</p>
<p class="boxes_labjournal_text">...first transduced cells...</p>
</a>
</a>
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<h2 style="margin-left: 5px;">July</h2>
<h2 style="margin-left: 5px;">July</h2>
<p class="boxes_labjournal_day">Labday 46- 75</p>
<p class="boxes_labjournal_day">Labday 46- 75</p>
-
<img src="http://2010.igem.org/wiki/images/3/35/Freiburg_10_BioBrick_icon.png" class="image_labjournal" />
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<img src="http://2010.igem.org/wiki/images/8/8d/Freiburg_10_labjournal_july_v1.JPG" class="image_labjournal" title="We developed a plan for modifying the surface exposed loops of the virus capsid.
 +
For this purpose not only 5 iGEM restriction sites, but also the so called ViralBrick restriction sites located in the Rep/Cap sequence needed to be deleted: BamHI, SalI. BioBrick production of the inverted terminal repeats (ITRs) turned out to be difficult. Therefore the alternative “ITR fancy method” was developed. We successfully produced AAV2 left and right ITR fused to the RFC10 prefix. Further on we received first quantitative transduction results via flow cytometry. mGMK_TK30 was successfully cloned into the vector plasmid." />
<p class="boxes_labjournal_text">...organizing the lab...</p>
<p class="boxes_labjournal_text">...organizing the lab...</p>
</a>
</a>
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<h2 style="margin-left: 5px;">August I</h2>
<h2 style="margin-left: 5px;">August I</h2>
<p class="boxes_labjournal_day">Labday 76- 92</p>
<p class="boxes_labjournal_day">Labday 76- 92</p>
-
<img src="http://2010.igem.org/wiki/images/3/35/Freiburg_10_BioBrick_icon.png" class="image_labjournal" />
+
<img src="http://2010.igem.org/wiki/images/8/8b/Freiburg_10_labjournal_august1_v1.JPG" class="image_labjournal" />
<p class="boxes_labjournal_text">...cloning parts...</p>
<p class="boxes_labjournal_text">...cloning parts...</p>
</a>
</a>
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<h2 style="margin-left: 5px;">August II</h2>
<h2 style="margin-left: 5px;">August II</h2>
<p class="boxes_labjournal_day">Labday 93- 106</p>
<p class="boxes_labjournal_day">Labday 93- 106</p>
-
<img src="http://2010.igem.org/wiki/images/3/35/Freiburg_10_BioBrick_icon.png" class="image_labjournal" />
+
<img src="http://2010.igem.org/wiki/images/e/ec/Freiburg_10_labjournal_august2_v1.png" class="image_labjournal" />
<p class="boxes_labjournal_text">...assembled vector plasmid is working...</p>
<p class="boxes_labjournal_text">...assembled vector plasmid is working...</p>
</a>
</a>
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<h2 style="margin-left: 5px;">September I</h2>
<h2 style="margin-left: 5px;">September I</h2>
<p class="boxes_labjournal_day">Labday 107- 123</p>
<p class="boxes_labjournal_day">Labday 107- 123</p>
-
<img src="http://2010.igem.org/wiki/images/3/35/Freiburg_10_BioBrick_icon.png" class="image_labjournal" />
+
<img src="http://2010.igem.org/wiki/images/2/25/Freiburg_10_labjournal_september1_v1.JPG" class="image_labjournal" />
<p class="boxes_labjournal_text">...simply producing BioBricks...</p>
<p class="boxes_labjournal_text">...simply producing BioBricks...</p>
</a>
</a>
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<h2 style="margin-left: 5px;">September II</h2>
<h2 style="margin-left: 5px;">September II</h2>
<p class="boxes_labjournal_day">Labday 124- 135</p>
<p class="boxes_labjournal_day">Labday 124- 135</p>
-
<img src="http://2010.igem.org/wiki/images/3/35/Freiburg_10_BioBrick_icon.png" class="image_labjournal" />
+
<img src="http://2010.igem.org/wiki/images/1/18/Freiburg_10_labjournal_october2_v1.JPG" class="image_labjournal" />
<p class="boxes_labjournal_text">...21x mutated RepCap is working...</p>
<p class="boxes_labjournal_text">...21x mutated RepCap is working...</p>
</a>
</a>
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<h2 style="margin-left: 5px;">October I</h2>
<h2 style="margin-left: 5px;">October I</h2>
<p class="boxes_labjournal_day">Labday 136- 149</p>
<p class="boxes_labjournal_day">Labday 136- 149</p>
-
<img src="http://2010.igem.org/wiki/images/3/35/Freiburg_10_BioBrick_icon.png" class="image_labjournal" />
+
<img src="http://2010.igem.org/wiki/images/e/e0/Freiburg_10_labjournal_october_v1.JPG" class="image_labjournal" />
<p class="boxes_labjournal_text">...testing BioBricks by virus production...</p>
<p class="boxes_labjournal_text">...testing BioBricks by virus production...</p>
</a>
</a>
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<h2 style="margin-left: 5px;">October II</h2>
<h2 style="margin-left: 5px;">October II</h2>
<p class="boxes_labjournal_day">Labday 150- 166</p>
<p class="boxes_labjournal_day">Labday 150- 166</p>
-
<img src="http://2010.igem.org/wiki/images/3/35/Freiburg_10_BioBrick_icon.png" class="image_labjournal" />
+
<img src="http://2010.igem.org/wiki/images/2/22/Freiburg10_Oktober.png" class="image_labjournal" />
<p class="boxes_labjournal_text">...Capsid modification works...</p>
<p class="boxes_labjournal_text">...Capsid modification works...</p>
</a>
</a>
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<h2 style="margin-left: 5px;">November</h2>
<h2 style="margin-left: 5px;">November</h2>
<p class="boxes_labjournal_day">Labday 166- 170</p>
<p class="boxes_labjournal_day">Labday 166- 170</p>
-
<img src="http://2010.igem.org/wiki/images/3/35/Freiburg_10_BioBrick_icon.png" class="image_labjournal" />
+
<img src="http://2010.igem.org/wiki/images/9/93/Freiburg10_November.png" class="image_labjournal" />
-
<p class="boxes_labjournal_text">...Jemboree!!!...</p>
+
<p class="boxes_labjournal_text">...Jamboree!!!...</p>
</a>
</a>
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<br><h2>Get to know our virus</h2>
 
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<!---Insert Text in here---> Our project starts with first sequence analysis.
 
</div>
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<div class="lab_journal_img"><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/March" ><img src="http://2010.igem.org/wiki/images/2/21/Freiburg10_Labjournal-March.png" /></a></div></div>
 
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<br><h2>Checking iGEM compatibility </h2>
 
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<!---Insert Text in here--->Scanning for iGEM restriction sites:<br/>
 
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• Vector plasmid <br/>
 
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• Thymidine kinase<br/>
 
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• Cytosindeaminase<br/>
 
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-
</div>
 
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<div class="lab_journal_img"><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/April" ><img src="http://2010.igem.org/wiki/images/6/6b/Freiburg10_Labjournal-April.png" /></a></div></div>
 
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<br><h2>First practical steps</h2>
 
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<!---Insert Text in here--->We took a closer look to the theoretical DNA- and amino acid sequence of the ITRs, questioned the role of the β-globin intron, had a closer look to the CMV promoter and performed our first cloning attempt.
 
-
</div>
 
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<div class="lab_journal_img"><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/May" ><img src="http://2010.igem.org/wiki/images/6/61/Freiburg10_Labjournal-May.png" /></a></div></div>
 
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<br><h2>First fluorescing HT1080 cells</h2>
 
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<!---Insert Text in here--->We conducted our first site-directed mutagenesis. <br/>
 
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<br/>
 
-
First cell culture steps:<br/>
 
-
• Splitting and seeding of AAV 293 and HT1080 cells<br/>
 
-
• Calcium phosphate transfection<br/>
 
-
•      Transduction with virus particles containing YFP encoding sequence<br/>
 
-
• Microscopy: Fluorescent transduced HT1080 cells<br/>
 
-
<br/>
 
-
We planned to determine infectious virus titer via quantitative real-time PCR. For this purpose primers were designed, transduced HT1080 cells were harvested and prepared.<br/>
 
-
<br/>
 
-
Theoretical studies of the AAV structure: Impressions.
 
-
 
-
</div>
 
-
<div class="lab_journal_img"><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/June" ><img src="http://2010.igem.org/wiki/images/4/48/Freiburg10_Labjournal-June.png" /></a></div></div>
 
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<div class="lab_journal_box">
 
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<br><h2>Modifying Rep/Cap & Modularizing Vector plasmid</h2>
 
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<!---Insert Text in here--->We developed a plan for modifying the surface exposed loops of the virus capsid.
 
-
For this purpose not only 5 iGEM restriction sites, but also the so called ViralBrick restriction sites located in the Rep/Cap sequence needed to be deleted: BamHI, SalI <br/>
 
-
<br/>
 
-
 
-
BioBrick production of the inverted terminal repeats (ITRs) turned out to be difficult. Therefore the alternative “ITR fancy method” was developed. We successfully produced AAV2 left and right ITR fused to the RFC10 prefix.<br/>
 
-
<br/>
 
-
Further on we received first quantitative transduction results via flow cytometry.<br/>
 
-
<br/>
 
-
mGMK_TK30 was successfully cloned into the vector plasmid.
 
-
 
-
</div>
 
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<div class="lab_journal_img"><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/July" ><img src="http://2010.igem.org/wiki/images/d/dc/Freiburg10_Labjournal-July.png" /></a></div></div>
 
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<br><h2>Producing more BioBricks</h2>
 
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http://2010.igem.org/Team:Freiburg_Bioware/NoteBook => Back to Notebook overview]
 
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<ul>
 
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/March">March (labday 1)</a></li>
 
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/April">April (labday 2 - 5)</a></li>
 
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/May">May (labday 6 - 17)</a></li>
 
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/June">June (labday 18 - 45)</a></li>
 
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/July">July (labday 46 - 75)</a></li>
 
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August">August part 1 (labday 76 - 92)</a></li>
 
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August2">August part 2 (labday 93 - 106)</a></li>
 
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September">September part 1 (labday 107 - 123)</a></li>
 
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September2">September part 2 (labday 124 - 135)</a></li>
 
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October">October part 1 (labday 136 - 149 )</a></li>
 
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October2">October part 2 (labday 150 - 166 )</a></li>
 
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November">November  (labday 167 - 170 )</a></li>
 
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/Cellculture">Cellculture</a></li>
 
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Latest revision as of 03:17, 28 October 2010