Team:KAIST Korea/Notebook Labnote/2012 7

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<section id="1">
<section id="1">
<div class="date">July 1<sup>st</sup> 2012</div></br>
<div class="date">July 1<sup>st</sup> 2012</div></br>
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<div class="note-title"><i>Moth 0109</i>, <i>1202</i> TOPO cloning colony PCR</div>
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<div id="content_note" >
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<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="0701Fig1" src="https://static.igem.org/mediawiki/2012/b/b9/KAIST_07012012_Fig1.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<span id="little">We did colony PCR for the 5 colony of TOPO cloned samples, but there was no band, which means TOPO cloning has failed for these two genes.</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
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<section id="2">
<section id="2">
<div class="date">July 2<sup>nd</sup> 2012</div></br>
<div class="date">July 2<sup>nd</sup> 2012</div></br>
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<div class="note-title">fdnG deletion PCP20 curing</div>
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</br><hr></br>
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<div class="note-title">fdnG deletion PCP20 prep, single cut</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
<section id="3">
<section id="3">
<div class="date">July 3<sup>rd</sup> 2012</div></br>
<div class="date">July 3<sup>rd</sup> 2012</div></br>
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<div class="note-title"><i>Moth_0109</i>, <i>1202</i> PCR</div>
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<div id="content_note" >
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<span id="little">As TOPO cloning of <i>Moth_0109</i> and <i>1202</i> gene failed, we ran one more PCR to prepare for TOPO cloning.</span>
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</br></br>
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<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="0703Fig1" src="https://static.igem.org/mediawiki/2012/e/e4/KAIST_07032012_Fig1.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<span id="little">For each of the two genes, correct size of oligonucleotide exists in the PCR product. We noticed that there is an other restriction enzyme site in the <i>Moth 0109</i>, <i>1202</i>. It means that it’s impossible to reuse TOPO vector. We got other vector which has same pBAD promoter with TOPO cloning. Also we redesigned the primer for <i>Moth 0109</i>, <i>1202</i>.</span>
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</br>
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</div>
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</br><hr></br>
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<div class="note-title">pre-culture of <i>Moth_1197</i> and <i>1198</i> inserted MG1655 cell. </div>
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<div id="content_note" >
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<span id="little">We did pre-culture of vector transformed cell into 3ml LB and incubated on 37℃ for 16 hours.</span>
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</br>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
<section id="4">
<section id="4">
<div class="date">July 4<sup>th</sup> 2012</div></br>
<div class="date">July 4<sup>th</sup> 2012</div></br>
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<div class="note-title">Induction of <i>Moth_1197</i>, <i>Moth_1198</i> gene (sampling) and running SDS-PAGE gel.</div>
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<div id="content_note" >
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<span id="little">We induced <i>Moth_1197</i> and <i>Moth_1198</i> gene in pTrcHis2A and pBAD vector with various IPTG and L-arabinose conditions: IPTG 0.5mM, 1mM and Arabinose 10mM, 30mM (0.02% and 0.2%). Temperature conditions are : 37℃, 30℃. Cells were cultured anaerobically in the anaerobic chamber. After 6 hours of induction, we run the sample into SDS-PAGE gel. The gel is stained with coomassie blue for 1 hour and de-stained for overnight.</span>
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</br></br>
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</div>
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<!--Anaerobic chamber 조성?-->
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
<section id="5">
<section id="5">
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<!--제목에 물음표는 뭘까;;?-->
<div class="date">July 5<sup>th</sup> 2012</div></br>
<div class="date">July 5<sup>th</sup> 2012</div></br>
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<div class="note-title">fdhF Knockout PCR ( negative and knockout) and PCP20?</div>
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<div id="content_note" >
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<b>Results</b></br></br>
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</div>
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</br><hr></br>
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<div class="note-title"><i>Moth_1197</i>-pBAD/TOPO expression check (Result)</div>
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<div id="content_note" >
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<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="0705Fig1" src="https://static.igem.org/mediawiki/2012/b/bb/KAIST_07052012_Fig1.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<b>Discussion</b></br></br>
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<span id="little">For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 28.61 kDa protein, Methyltetrahydrofolate : corrinoid/iron sulfur protein methyltransferase. The band significantly appeared slightly above 42kDa. This means that protein has N and C terminal tag, of which the size is 13kDa and 3kDa each.</br></br>
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<div align="center">28.61kDa + N-term Thioredoxin 13kDa + C-term V5, 6xHis 3kDa = 44.61kDa</div></br>
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Therefore, the product protein locates slightly above 42kDa ladder.</span>
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</br>
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</div>
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</br><hr></br>
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<div class="note-title"><i>Moth_1197</i>-pTrcHis2A expression check (Result)</div>
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<div id="content_note" >
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<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="0705Fig2" src="https://static.igem.org/mediawiki/2012/c/c4/KAIST_07052012_Fig2.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<b>Discussion</b></br></br>
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<span id="little">For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 28.61 kDa protein, Methyltetrahydrofolate : corrinoid/iron sulfur protein methyltransferase. The band significantly appeared between 24kDa and 35kDa. This means that protein has C terminal tag, </br></br>
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<div align="center">28.61kDa + 2.14kDa = 30.75kDa</div></br>
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The band that does not exist on negative sample is roughly on this size. Therefore, we can say that this protein is also expressed on pTrcHis2A vector. However, we decided to use pBAD/TOPO vector because it has expressed the protein more significantly.</span>
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</br>
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</div>
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</br><hr></br>
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<div class="note-title"><i>Moth_1198</i>-pBAD/TOPO expression check (Result)</div>
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<div id="content_note" >
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<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="0705Fig3" src="https://static.igem.org/mediawiki/2012/c/cf/KAIST_07052012_Fig3.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<b>Discussion</b></br></br>
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<span id="little">For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 35.08kDa protein acetyl-CoA decarbonylase/synthase complex subunit delta. But the band was not certain on the gel image.</span>
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</br>
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</div>
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</br><hr></br>
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<div class="note-title"><i>Moth_1198</i>-pTrcHis2A expression check (Result)</div>
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<div id="content_note" >
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<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="0705Fig4" src="https://static.igem.org/mediawiki/2012/c/c1/KAIST_07052012_Fig4.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<b>Discussion</b></br></br>
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<span id="little">For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 35.08kDa protein acetyl-CoA decarbonylase/synthase complex subunit delta. The band of correct size appeared  on the soluble fraction of each sample.</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
<section id="6">
<section id="6">
<div class="date">July 6<sup>th</sup> 2012</div></br>
<div class="date">July 6<sup>th</sup> 2012</div></br>
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<div id="content_note" >
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<span id="little">No Special Event!</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
<section id="7">
<section id="7">
<div class="date">July 7<sup>th</sup> 2012</div></br>
<div class="date">July 7<sup>th</sup> 2012</div></br>
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<div id="content_note" >
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<span id="little">No Special Event!</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
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<section id="8">
<section id="8">
<div class="date">July 8<sup>th</sup> 2012</div></br>
<div class="date">July 8<sup>th</sup> 2012</div></br>
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<div class="note-title"><i>Moth 0109</i>, <i>1202</i> colony PCR for TOPO cloning to TOP10</div>
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<div id="content_note" >
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<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="0708Fig1" src="https://static.igem.org/mediawiki/2012/3/3f/KAIST_07082012_Fig1.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<span id="little">1202 colony PCR, The size of bands are correct. We succeed <i>Moth 0109</i>, <i>1202</i> gene cloning.</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
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<section id="9">
<section id="9">
<div class="date">July 9<sup>th</sup> 2012</div></br>
<div class="date">July 9<sup>th</sup> 2012</div></br>
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<div class="note-title">fdoG knockout confirm PCR</div>
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<div id="content_note" >
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<b>Results</b></br></br>
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</div>
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</br><hr></br>
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<div class="note-title"><i>Moth 0109</i> and <i>Moth 1202</i> electro transformation into MG1655 cell</div>
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<div id="content_note" >
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<b>Procedure</b></br></br>
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<span id="little">We did mini-prep to get the vectors which has <i>Moth 0109</i>, <i>1202</i> each from TOP10. We did electroporation to MG1655.</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
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<section id="10">
<section id="10">
<div class="date">July 10<sup>th</sup> 2012</div></br>
<div class="date">July 10<sup>th</sup> 2012</div></br>
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<div class="note-title">piBR181 vector arrived</div>
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<div id="content_note" >
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<span id="little">piBR181 vector is known to accommodate oligonucleotide over 100kb. This vector was made from commercial pET-28a (+) vectors to accommodate standardized Biobricks parts. The work proceeded by designing a 181 bp long synthetic DNA fragment for efficient construction of an expression vector for monocistronic assembly of genes provided that each contains its own promoter, operator, ribosome-binding site and transcriptional terminator. A 181 bp long ds DNA construct was designed and ligated to pET-28a (+) (5369 bp) at BglII and XhoI restriction sites to generate piBR181 (5301 bp) expression vector replacing original bio-parts. The newly constructed vector contains SpeI-HindIII single cloning restriction site between RBS and TT sites however, BglII and BamHI/XhoI restriction sites are present before promoter and after TT sites for multi-monocistronic operon assembly.</span>
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</br></br>
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<div align="center"><img id="figure" alt="0710Fig1" src="https://static.igem.org/mediawiki/2012/4/4c/KAIST_07102012_Fig1.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<span id="little">We got this standardized vector and vector map from Professor Jae Kyung, Song and it arrived today. </span>
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</br></br>
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<span id="little"><a href="#">▶▶Click to download Original document of piBR181 vector</a></span>
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<!--벡터 맵 워드파일로 첨부할것-->
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</div>
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</br><hr></br>
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<div class="note-title"><i>Moth 0109</i> transformed cell confirm PCR</div>
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<div id="content_note" >
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<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="0710Fig2" src="https://static.igem.org/mediawiki/2012/8/8f/KAIST_07102012_Fig2.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<span id="little">There was no band appeared on the gel, which means that we don’t have any colony with <i>Moth 0109</i> gene.</span>
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</br>
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</div>
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</br><hr></br>
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<div class="note-title"><i>Moth 1202</i> transformed cell confirm PCR</div>
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<div id="content_note" >
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<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="0710Fig3" src="https://static.igem.org/mediawiki/2012/3/35/KAIST_07102012_Fig3.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<span id="little">Band size is smaller than <i>Moth 1202</i>. It means we failed to transform <i>Moth 1202</i> gene in to MG1655.</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
<section id="11">
<section id="11">
<div class="date">July 11<sup>th</sup> 2012</div></br>
<div class="date">July 11<sup>th</sup> 2012</div></br>
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<div id="content_note" >
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<span id="little">No Special Event!</span>
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</br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
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<section id="12">
<section id="12">
<div class="date">July 12<sup>th</sup> 2012</div></br>
<div class="date">July 12<sup>th</sup> 2012</div></br>
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<div class="note-title">pre-culture of <i>Moth_1202</i>, <i>2312</i> and <i>2315</i> inserted MG1655 cell. </div>
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<div id="content_note" >
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<span id="little">We did pre-culture of vector transformed cell into 3ml LB and incubated on 37℃ for 16 hours. </span>
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</br></br>
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</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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</section>
</section>
<section id="13">
<section id="13">
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<div class="date">July 15<sup>th</sup> 2012</div></br>
<div class="date">July 15<sup>th</sup> 2012</div></br>
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<div class="date">July 16<sup>th</sup> 2012</div></br>
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<div class="date">July 17<sup>th</sup> 2012</div></br>
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<div class="date">July 18<sup>th</sup> 2012</div></br>
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<div class="date">July 19<sup>th</sup> 2012</div></br>
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<div class="date">July 20<sup>th</sup> 2012</div></br>
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<div class="date">July 21<sup>st</sup> 2012</div></br>
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<div class="date">July 22<sup>nd</sup> 2012</div></br>
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<div class="date">July 23<sup>rd</sup> 2012</div></br>
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<div class="date">July 24<sup>th</sup> 2012</div></br>
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<div class="date">July 25<sup>th</sup> 2012</div></br>
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<div class="date">July 26<sup>th</sup> 2012</div></br>
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<div class="date">July 27<sup>th</sup> 2012</div></br>
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<div class="date">July 28<sup>th</sup> 2012</div></br>
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<div class="date">July 29<sup>th</sup> 2012</div></br>
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<div class="date">July 30<sup>th</sup> 2012</div></br>
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Revision as of 13:29, 10 September 2012

KAIST Korea 2012 iGEM

Notebook : Labnote-July

Labnote

July

July 1st 2012

Moth 0109, 1202 TOPO cloning colony PCR
Results

0701Fig1

We did colony PCR for the 5 colony of TOPO cloned samples, but there was no band, which means TOPO cloning has failed for these two genes.
Back to the Calendar
July 2nd 2012

fdnG deletion PCP20 curing


fdnG deletion PCP20 prep, single cut
Back to the Calendar
July 3rd 2012

Moth_0109, 1202 PCR
As TOPO cloning of Moth_0109 and 1202 gene failed, we ran one more PCR to prepare for TOPO cloning.

Results

0703Fig1

For each of the two genes, correct size of oligonucleotide exists in the PCR product. We noticed that there is an other restriction enzyme site in the Moth 0109, 1202. It means that it’s impossible to reuse TOPO vector. We got other vector which has same pBAD promoter with TOPO cloning. Also we redesigned the primer for Moth 0109, 1202.


pre-culture of Moth_1197 and 1198 inserted MG1655 cell.
We did pre-culture of vector transformed cell into 3ml LB and incubated on 37℃ for 16 hours.

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July 4th 2012

Induction of Moth_1197, Moth_1198 gene (sampling) and running SDS-PAGE gel.
We induced Moth_1197 and Moth_1198 gene in pTrcHis2A and pBAD vector with various IPTG and L-arabinose conditions: IPTG 0.5mM, 1mM and Arabinose 10mM, 30mM (0.02% and 0.2%). Temperature conditions are : 37℃, 30℃. Cells were cultured anaerobically in the anaerobic chamber. After 6 hours of induction, we run the sample into SDS-PAGE gel. The gel is stained with coomassie blue for 1 hour and de-stained for overnight.

Back to the Calendar
July 5th 2012

fdhF Knockout PCR ( negative and knockout) and PCP20?
Results



Moth_1197-pBAD/TOPO expression check (Result)
Results

0705Fig1

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 28.61 kDa protein, Methyltetrahydrofolate : corrinoid/iron sulfur protein methyltransferase. The band significantly appeared slightly above 42kDa. This means that protein has N and C terminal tag, of which the size is 13kDa and 3kDa each.

28.61kDa + N-term Thioredoxin 13kDa + C-term V5, 6xHis 3kDa = 44.61kDa

Therefore, the product protein locates slightly above 42kDa ladder.


Moth_1197-pTrcHis2A expression check (Result)
Results

0705Fig2

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 28.61 kDa protein, Methyltetrahydrofolate : corrinoid/iron sulfur protein methyltransferase. The band significantly appeared between 24kDa and 35kDa. This means that protein has C terminal tag,

28.61kDa + 2.14kDa = 30.75kDa

The band that does not exist on negative sample is roughly on this size. Therefore, we can say that this protein is also expressed on pTrcHis2A vector. However, we decided to use pBAD/TOPO vector because it has expressed the protein more significantly.


Moth_1198-pBAD/TOPO expression check (Result)
Results

0705Fig3

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 35.08kDa protein acetyl-CoA decarbonylase/synthase complex subunit delta. But the band was not certain on the gel image.


Moth_1198-pTrcHis2A expression check (Result)
Results

0705Fig4

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 35.08kDa protein acetyl-CoA decarbonylase/synthase complex subunit delta. The band of correct size appeared on the soluble fraction of each sample.
Back to the Calendar
July 6th 2012

No Special Event!
Back to the Calendar
July 7th 2012

No Special Event!
Back to the Calendar
July 8th 2012

Moth 0109, 1202 colony PCR for TOPO cloning to TOP10
Results

0708Fig1

1202 colony PCR, The size of bands are correct. We succeed Moth 0109, 1202 gene cloning.
Back to the Calendar
July 9th 2012

fdoG knockout confirm PCR
Results



Moth 0109 and Moth 1202 electro transformation into MG1655 cell
Procedure

We did mini-prep to get the vectors which has Moth 0109, 1202 each from TOP10. We did electroporation to MG1655.
Back to the Calendar
July 10th 2012

piBR181 vector arrived
piBR181 vector is known to accommodate oligonucleotide over 100kb. This vector was made from commercial pET-28a (+) vectors to accommodate standardized Biobricks parts. The work proceeded by designing a 181 bp long synthetic DNA fragment for efficient construction of an expression vector for monocistronic assembly of genes provided that each contains its own promoter, operator, ribosome-binding site and transcriptional terminator. A 181 bp long ds DNA construct was designed and ligated to pET-28a (+) (5369 bp) at BglII and XhoI restriction sites to generate piBR181 (5301 bp) expression vector replacing original bio-parts. The newly constructed vector contains SpeI-HindIII single cloning restriction site between RBS and TT sites however, BglII and BamHI/XhoI restriction sites are present before promoter and after TT sites for multi-monocistronic operon assembly.

0710Fig1

We got this standardized vector and vector map from Professor Jae Kyung, Song and it arrived today.

▶▶Click to download Original document of piBR181 vector


Moth 0109 transformed cell confirm PCR
Results

0710Fig2

There was no band appeared on the gel, which means that we don’t have any colony with Moth 0109 gene.


Moth 1202 transformed cell confirm PCR
Results

0710Fig3

Band size is smaller than Moth 1202. It means we failed to transform Moth 1202 gene in to MG1655.
Back to the Calendar
July 11th 2012

No Special Event!
Back to the Calendar
July 12th 2012

pre-culture of Moth_1202, 2312 and 2315 inserted MG1655 cell.
We did pre-culture of vector transformed cell into 3ml LB and incubated on 37℃ for 16 hours.

Back to the Calendar
July 13th 2012

July 14th 2012

July 15th 2012



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