Team:KAIST Korea/Notebook Labnote/2012 8

From 2012.igem.org

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<section id="1">
<section id="1">
<div class="date">August 1<sup>st</sup> 2012</div></br>
<div class="date">August 1<sup>st</sup> 2012</div></br>
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:262px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="packman" src='http://2012.igem.org/wiki/images/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></div></br>
<div class="note-title">PCR amplification of <i>Moth_1191</i>, <i>1199</i> gene : decreasing annealing temperature down to 45℃</div>
<div class="note-title">PCR amplification of <i>Moth_1191</i>, <i>1199</i> gene : decreasing annealing temperature down to 45℃</div>
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</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
<div class="note-title">Induction of <i>Moth_0109</i> gene (sampling) and running SDS-PAGE gel. </div>
<div class="note-title">Induction of <i>Moth_0109</i> gene (sampling) and running SDS-PAGE gel. </div>
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</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
<div class="note-title">PCR amplification of <i>Moth_1191</i>, <i>1199</i> gene with Gibson and TOPO primer</div>
<div class="note-title">PCR amplification of <i>Moth_1191</i>, <i>1199</i> gene with Gibson and TOPO primer</div>
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<section id="2">
<section id="2">
<div class="date">August 2<sup>nd</sup> 2012</div></br>
<div class="date">August 2<sup>nd</sup> 2012</div></br>
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:262px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="packman" src='http://2012.igem.org/wiki/images/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></div></br>
<div class="note-title">Primer design for FDH derived from <i>Candida boidinii</i> mutant</div>
<div class="note-title">Primer design for FDH derived from <i>Candida boidinii</i> mutant</div>
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</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
<div class="note-title">Expression check of <i>Moth_0109</i> gene on pBAD/mycHisC vector (Result)</div>
<div class="note-title">Expression check of <i>Moth_0109</i> gene on pBAD/mycHisC vector (Result)</div>
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<section id="4">
<section id="4">
<div class="date">August 4<sup>th</sup> 2012</div></br>
<div class="date">August 4<sup>th</sup> 2012</div></br>
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:262px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="packman" src='http://2012.igem.org/wiki/images/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></div></br>
<div class="note-title"><i>1191</i> and <i>1199</i> forward primer design</div>
<div class="note-title"><i>1191</i> and <i>1199</i> forward primer design</div>
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<section id="7">
<section id="7">
<div class="date">August 7<sup>th</sup> 2012</div></br>
<div class="date">August 7<sup>th</sup> 2012</div></br>
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:262px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="packman" src='http://2012.igem.org/wiki/images/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></div></br>
<div class="note-title">FDH gene PCR amplification</div>
<div class="note-title">FDH gene PCR amplification</div>
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<section id="8">
<section id="8">
<div class="date">August 8<sup>th</sup> 2012</div></br>
<div class="date">August 8<sup>th</sup> 2012</div></br>
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:262px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="packman" src='http://2012.igem.org/wiki/images/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></div></br>
<div class="note-title">FDH gene and pTrcHis2A, pBAD/mycHisC vector enzyme digestion</div>
<div class="note-title">FDH gene and pTrcHis2A, pBAD/mycHisC vector enzyme digestion</div>
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</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
<div class="note-title">pTrcHis2A, pBAD/myc vector dephosphorylation</div>
<div class="note-title">pTrcHis2A, pBAD/myc vector dephosphorylation</div>
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</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
<div class="note-title">ligation of FDH gene with pTrcHis2A and pBAD/mycHisC vectors</div>
<div class="note-title">ligation of FDH gene with pTrcHis2A and pBAD/mycHisC vectors</div>
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</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
<div class="note-title">Primer amplification of <i>1191</i> and <i>1199</i></div>
<div class="note-title">Primer amplification of <i>1191</i> and <i>1199</i></div>
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<section id="9">
<section id="9">
<div class="date">August 9<sup>th</sup> 2012</div></br>
<div class="date">August 9<sup>th</sup> 2012</div></br>
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:262px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="packman" src='http://2012.igem.org/wiki/images/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></div></br>
<div class="note-title">vector transformation into TOP10 cell</div>
<div class="note-title">vector transformation into TOP10 cell</div>
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<section id="11">
<section id="11">
<div class="date">August 11<sup>th</sup> 2012</div></br>
<div class="date">August 11<sup>th</sup> 2012</div></br>
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:262px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="packman" src='http://2012.igem.org/wiki/images/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></div></br>
<div class="note-title">pTrcHis2A vector transformation into <i>E.coli</i> MG1655 cell</div>
<div class="note-title">pTrcHis2A vector transformation into <i>E.coli</i> MG1655 cell</div>
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</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
<div class="note-title">pBAD/mycHisC-FDH transformation into TOP10</div>
<div class="note-title">pBAD/mycHisC-FDH transformation into TOP10</div>
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<section id="12">
<section id="12">
<div class="date">August 12<sup>th</sup> 2012</div></br>
<div class="date">August 12<sup>th</sup> 2012</div></br>
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:262px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="packman" src='http://2012.igem.org/wiki/images/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></div></br>
<div class="note-title">colony PCR check of pTrcHis2A inserted FDH gene</div>
<div class="note-title">colony PCR check of pTrcHis2A inserted FDH gene</div>
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</div>
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</br><hr></br>
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</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
<div class="note-title">pBAD FDH MG1655 electroporation</div>
<div class="note-title">pBAD FDH MG1655 electroporation</div>
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<section id="13">
<section id="13">
<div class="date">August 13<sup>th</sup> 2012</div></br>
<div class="date">August 13<sup>th</sup> 2012</div></br>
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:262px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="packman" src='http://2012.igem.org/wiki/images/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></div></br>
<div class="note-title">FDH pBAD MG1655 electroporation</div>
<div class="note-title">FDH pBAD MG1655 electroporation</div>
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</div>
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</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
<div class="note-title">FDH pTrc MG1655 expression check (sampling and induction)</div>
<div class="note-title">FDH pTrc MG1655 expression check (sampling and induction)</div>
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<section id="14">
<section id="14">
<div class="date">August 14<sup>th</sup> 2012</div></br>
<div class="date">August 14<sup>th</sup> 2012</div></br>
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:262px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="packman" src='http://2012.igem.org/wiki/images/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></div></br>
<div class="note-title">colony PCR check of pBADmycHisC inserted FDH gene</div>
<div class="note-title">colony PCR check of pBADmycHisC inserted FDH gene</div>
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</div>
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</br><hr></br>
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</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
<div class="note-title">Expression check of Formate dehydrogenase on pTrcHis2A vector (Result)</div>
<div class="note-title">Expression check of Formate dehydrogenase on pTrcHis2A vector (Result)</div>
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</br><hr></br>
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</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
<div class="note-title">FDH pBAD MG1655 seeding for expression check</div>
<div class="note-title">FDH pBAD MG1655 seeding for expression check</div>
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</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
<div class="note-title">Primer design for the insertion of <i>1202</i> and <i>0109</i></div>
<div class="note-title">Primer design for the insertion of <i>1202</i> and <i>0109</i></div>
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<section id="15">
<section id="15">
<div class="date">August 15<sup>th</sup> 2012</div></br>
<div class="date">August 15<sup>th</sup> 2012</div></br>
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:262px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="packman" src='http://2012.igem.org/wiki/images/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></div></br>
<div class="note-title">FDH pBAD MG1655 expression check</div>
<div class="note-title">FDH pBAD MG1655 expression check</div>
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<section id="16">
<section id="16">
<div class="date">August 16<sup>th</sup> 2012</div></br>
<div class="date">August 16<sup>th</sup> 2012</div></br>
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:262px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="packman" src='http://2012.igem.org/wiki/images/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></div></br>
<div class="note-title">PCR amplification of <i>1201</i>, <i>1202</i>, <i>1203</i>, <i>1198</i> and <i>1191</i></div>
<div class="note-title">PCR amplification of <i>1201</i>, <i>1202</i>, <i>1203</i>, <i>1198</i> and <i>1191</i></div>
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</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
<div class="note-title">Pre-culture of <i>0109</i></div>
<div class="note-title">Pre-culture of <i>0109</i></div>
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</br><div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='http://2012.igem.org/wiki/images/2/22/KAIST_FlipFlop.png'/> Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></div></br>
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<div class="note-title">pPoC and pPoCpi electro-transformation into MG1655</div>
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<div id="content_note" >
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<span>Strain: MG1655</br></br>
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Plasmid source:</br></br>
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<ul style="list-style-type:square">
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<li>pPoC: from DH5α-pPoC colony #1 and TOP10-pPoC colony #1</li>
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<li>pPoCpi: from TOP10-pPoC colony #1 and #3</li>
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</ul>
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</br></br>
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</span>
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</div>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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<section id="17">
<section id="17">
<div class="date">August 17<sup>th</sup> 2012</div></br>
<div class="date">August 17<sup>th</sup> 2012</div></br>
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:262px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="packman" src='http://2012.igem.org/wiki/images/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></div></br>
<div class="note-title">Expression check of Formate dehydrogenase.</div>
<div class="note-title">Expression check of Formate dehydrogenase.</div>
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</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
<div class="note-title">PCR amplification – second trial</div>
<div class="note-title">PCR amplification – second trial</div>
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</br>
</br>
</div>
</div>
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</br><div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='http://2012.igem.org/wiki/images/2/22/KAIST_FlipFlop.png'/> Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></div></br>
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<div class="note-title">MG1655-pPoC, MG1655-pPoCpi streaking</div>
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<div id="content_note" >
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<span id="little">We could not distinguish single colony from plates. Therefore we scraped cells and streaked onto new plates.</span>
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</br></br>
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<b>Discussions</b></br></br>
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<span id="little">Wild-type <i>E.coli</i> showed more reddish and yellowish color than competent strains, DH5α and TOP10. The promoter BBa_J23119 may show different strength in-between strains.</span>
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</br>
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</div>
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</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
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<div class="note-title">TOP10-pTrcHis2A_Bxb1 expression check </div>
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<div id="content_note" >
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<span id="little">TOP10 containing pTrcHis2A was cultured in 30mL of LB with 1% of Ampicillin.</br></span>
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<span id="little">IPTG induced when OD = 0.4</br></span>
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<span id="little">Sampling: 8 hours after induction, 1010 cells.</br></span>
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<span id="little">Only soluble fractions were PAGEed.</br></span>
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</br>
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</div>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
</section>
</section>
<section id="18">
<section id="18">
<div class="date">August 18<sup>th</sup> 2012</div></br>
<div class="date">August 18<sup>th</sup> 2012</div></br>
-
 
+
<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:262px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="packman" src='http://2012.igem.org/wiki/images/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></div></br>
<div class="note-title">PCR amplification of <i>1202</i>, <i>0109</i> gene with original primer and Gibson primers</div>
<div class="note-title">PCR amplification of <i>1202</i>, <i>0109</i> gene with original primer and Gibson primers</div>
Line 807: Line 855:
</div>
</div>
-
</br><hr></br>
+
</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
<div class="note-title"><i>1202</i> and <i>0109</i> forward primer design</div>
<div class="note-title"><i>1202</i> and <i>0109</i> forward primer design</div>
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<!--여기부터 없음-->
<!--여기부터 없음-->
</div>
</div>
 +
</br><div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='http://2012.igem.org/wiki/images/2/22/KAIST_FlipFlop.png'/> Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></div></br>
 +
 +
<div class="note-title">MG1655-pPoC, MG1655-pPoCpi colony inoculation</div>
 +
<div id="content_note" >
 +
 +
<span id="little">Two colonies from each plate were inoculated into 3mL of LB containing 1% of chloramphenicol.</span>
 +
</br></br>
 +
<b>Discussions</b></br></br>
 +
 +
 +
<span id="little">In 12 hours, cell cultures show colors compared to another common <i>E.coli</i> culture. When they are centrifuged, pellet shows clear and definite yellow or red color.</span>
 +
 +
</br>
 +
 +
 +
</div>
 +
 +
</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
 +
 +
<div class="note-title">pTrcHis2A_Bxb1 electro-transformation into MG1655-pPoC, MG1655-pPoCpi</div>
 +
 +
<div id="content_note" >
 +
 +
<span id="little">pTrcHis2A_Bxb1 is electro-transformed into MG1655 containing pPoC and pPoCpi.</br></br>
 +
  Plasmid source: TOP10-pTrcHis2A_Bxb1</span>
 +
</br></br>
 +
</div>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
Line 826: Line 901:
<section id="19">
<section id="19">
<div class="date">August 19<sup>th</sup> 2012</div></br>
<div class="date">August 19<sup>th</sup> 2012</div></br>
 +
<div id="content_note" >
 +
<span id="little">No Special Event!</span>
 +
</br>
 +
 +
</div>
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
</section>
</section>
<section id="20">
<section id="20">
<div class="date">August 20<sup>th</sup> 2012</div></br>
<div class="date">August 20<sup>th</sup> 2012</div></br>
 +
<div id="content_note" >
 +
<span id="little">No Special Event!</span>
 +
</br>
 +
 +
</div>
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
</section>
</section>
<section id="21">
<section id="21">
<div class="date">August 21<sup>st</sup> 2012</div></br>
<div class="date">August 21<sup>st</sup> 2012</div></br>
 +
<div id="content_note" >
 +
<span id="little">No Special Event!</span>
 +
</br>
 +
 +
</div>
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
</section>
</section>
<section id="22">
<section id="22">
<div class="date">August 22<sup>nd</sup> 2012</div></br>
<div class="date">August 22<sup>nd</sup> 2012</div></br>
 +
<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='http://2012.igem.org/wiki/images/2/22/KAIST_FlipFlop.png'/> Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></div></br>
 +
 +
<div class="note-title">Primer design for Bxb1 integrase cloning</div>
 +
 +
<div id="content_note" >
 +
 +
<span id="little">Primers for Bxb1 integrase cloning were designed. For controlling basal transcription level optimization, a mutant primer which has GTG rather than ATG for its start codon was also ordered.</span>
 +
</br></br>
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="2_0822Fig1" src="http://2012.igem.org/wiki/images/9/92/KAIST_2nd_08222012_Fig1.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
 +
</div>
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
</section>
</section>
<section id="23">
<section id="23">
<div class="date">August 23<sup>rd</sup> 2012</div></br>
<div class="date">August 23<sup>rd</sup> 2012</div></br>
 +
<div id="content_note" >
 +
<span id="little">No Special Event!</span>
 +
</br>
 +
 +
</div>
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
</section>
</section>
<section id="24">
<section id="24">
<div class="date">August 24<sup>th</sup> 2012</div></br>
<div class="date">August 24<sup>th</sup> 2012</div></br>
 +
<div id="content_note" >
 +
<span id="little">No Special Event!</span>
 +
</br>
 +
 +
</div>
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
</section>
</section>
<section id="25">
<section id="25">
<div class="date">August 25<sup>th</sup> 2012</div></br>
<div class="date">August 25<sup>th</sup> 2012</div></br>
 +
<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='http://2012.igem.org/wiki/images/2/22/KAIST_FlipFlop.png'/> Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></div></br>
 +
 +
<div class="note-title">pPoC-Bxb1 double transformants inoculation for prep & PCR check</div>
 +
 +
<div id="content_note" >
 +
 +
<span id="little">Double transformants 1 through 24 were inoculated into 3ml of LB containing 1% of Cm and Ap. For our convenience, three colonies were inoculated in one culture tube.</span>
 +
</br></br>
 +
 +
</div>
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
</section>
</section>
 +
<section id="26">
<section id="26">
<div class="date">August 26<sup>th</sup> 2012</div></br>
<div class="date">August 26<sup>th</sup> 2012</div></br>
 +
 +
</br><div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='http://2012.igem.org/wiki/images/2/22/KAIST_FlipFlop.png'/> Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></div></br>
 +
 +
<div class="note-title">pPoC-Bxb1 double transformants plasmid DNA miniprep & PCR check</div>
 +
 +
<div id="content_note" >
 +
 +
<span id="little">Inoculated cultures were mipi-preped. And Bxb1-pTrc plasmid was checked by PCR. In case of pPoC, it would be check by color.</span>
 +
</br></br>
 +
<b>Results</b></br></br>
 +
<span id="little">Vector</span></br></br>
 +
<div align="center"><img id="figure" alt="2_0826Fig1" src="http://2012.igem.org/wiki/images/3/38/KAIST_2nd_08252012_Fig1.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
<span id="little">Confirm PCR</span></br></br>
 +
<div align="center"><img id="figure" alt="2_0826Fig2" src="http://2012.igem.org/wiki/images/b/b9/KAIST_2nd_08252012_Fig2.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br></br>
 +
<b>Discussions</b></br></br>
 +
<span id="little">In confirm PCR, all the colonies seem to have Bxb1 vector. Colony #1, 4, 7, 10, 13, 14, 16, 19 and 22 will be inoculated.</span></br>
 +
 +
 +
</div>
 +
 +
</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
 +
 +
<div class="note-title">Bxb1 PCR with new primers</div>
 +
 +
<div id="content_note" >
 +
 +
<span id="little">For cloning of Bxb1 into pBAD and mutating its start codon from ATG to GTG, we PCR pGEM-B1-Bxb1 vector as template. Samples were duplicated</br></br></span>
 +
<span>
 +
PCR conditions
 +
<ol>
 +
<li>95℃ for 3min</li>
 +
<li>95℃ for 30sec</li>
 +
<li>55℃ for 30sec</li>
 +
<li>72℃ for 1min 30sec</li>
 +
Step 2 to 4: 34 cycles
 +
<li>72℃ for 10min</li>
 +
<li>12℃ forever</li>
 +
</ol>
 +
</span>
 +
</br></br>
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="2_0826Fig3" src="http://2012.igem.org/wiki/images/6/61/KAIST_2nd_08262012_Fig3.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
 +
<span id="little">Duplicated samples were purified in one DNA binding column.</br>ATG: 297.9ng/uL (purity: 1.90)</br>GTG: purify failed. Sample lost</br></span>
 +
 +
</br>
 +
<b>Discussions</b></br></br>
 +
<span id="little">There were multiple bands in our PCR products. We will PCR again Bxb1-GTG with different conditions. Template vector may show larger bands.</span>
 +
</br>
 +
 +
</div>
 +
</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
 +
 +
<div class="note-title">pGEM-Bxb1 template vector gel electrophoresis</div>
 +
 +
<div id="content_note" >
 +
 +
<span id="little">To check whether the undesired bands were template vector, we diluted template vector with same conditions of PCR, 50X, and electrophoresized.</span>
 +
</br></br>
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="2_0826Fig4" src="http://2012.igem.org/wiki/images/e/ea/KAIST_2nd_08262012_Fig4.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br></br>
 +
<b>Discussion</b></br></br>
 +
 +
<span id="little">The band was different from undesired bands from Bxb1 ATG, GTG PCR products.</span>
 +
 +
</br>
 +
</div>
 +
</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
 +
 +
<div class="note-title">Insert DNA (Bxb1 integrase) and vector (pTrc and pBAD) cloning</div>
 +
 +
<div id="content_note" >
 +
 +
<span id="little"></span>
 +
</div>
 +
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
</section>
</section>
 +
<section id="27">
<section id="27">
<div class="date">August 27<sup>th</sup> 2012</div></br>
<div class="date">August 27<sup>th</sup> 2012</div></br>
 +
</br><div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='http://2012.igem.org/wiki/images/2/22/KAIST_FlipFlop.png'/> Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></div></br>
 +
 +
<div class="note-title">Bxb1-pTrc and Bxb1-pBAD electrotransformation into MG1655</div>
 +
 +
<div id="content_note" >
 +
 +
</div>
 +
 +
</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
 +
 +
<div class="note-title">Bxb1 PCR with changed conditions</div>
 +
 +
<div id="content_note" >
 +
 +
<span id="little">For cloning of Bxb1 into pBAD and mutating its start codon from ATG to GTG, we PCR pGEM-B1-Bxb1 vector as template. Samples were duplicated</span>
 +
</br></br>
 +
<div align="center">
 +
<table style="border:0px;background-color:transparent;">
 +
<tr>
 +
<td style="padding:5px 20px 5px 20px;"><b>PCR conditions #1</b></td> <td style="padding:5px 20px 5px 20px;"><b>PCR conditions #2</b></td>
 +
</tr>
 +
<tr>
 +
<td style="padding:5px 20px 5px 20px;">1) 95℃ for 3min</td> <td style="padding:5px 20px 5px 20px;">1) 95℃ for 3min</td>
 +
</tr>
 +
<tr>
 +
<td style="padding:5px 20px 5px 20px;">2) 95℃ for 30sec</td> <td style="padding:5px 20px 5px 20px;">2) 95℃ for 30sec</td>
 +
</tr>
 +
<tr>
 +
<td style="padding:5px 20px 5px 20px;">3) 56.5℃ for 30sec</td> <td style="padding:5px 20px 5px 20px;">3) 58℃ for 30sec</td>
 +
</tr>
 +
<tr>
 +
<td style="padding:5px 20px 5px 20px;">4) 72℃ for 1min 30sec</br>  (Step 2 to 4: 34 cycles)</td> <td style="padding:5px 20px 5px 20px;">4) 72℃ for 1min 30sec</br>  (Step 2 to 4: 34 cycles)</td>
 +
</tr>
 +
<tr>
 +
<td style="padding:5px 20px 5px 20px;">5) 72℃ for 10min</td> <td style="padding:5px 20px 5px 20px;">5) 72℃ for 10min</td>
 +
</tr>
 +
<tr>
 +
<td style="padding:5px 20px 5px 20px;">6) 12℃ forever </td> <td style="padding:5px 20px 5px 20px;">6) 12℃ forever</td>
 +
</tr>
 +
 +
</table>
 +
 +
</div>
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="2_0827Fig1" src="http://2012.igem.org/wiki/images/a/a5/KAIST_2nd_08272012_Fig1.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br></br>
 +
<b>Discussions</b></br></br>
 +
 +
<span id="little">Still, undesired bands appeared. We do not know where they come from. We decide to extract desired DNA with gel extraction.</span>
 +
 +
</br>
 +
</div>
 +
</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
 +
 +
<div class="note-title">Bxb1, with GTG as start codon, PCR product gel extraction</div>
 +
 +
<div id="content_note" >
 +
 +
<span id="little">PCR products were gel extracted.</span>
 +
</br></br>
 +
<b>Results</b></br></br>
 +
<span id="little">Bxb1 GTG cond. #1 - #1: 128.2ng/uL (purity: 1.89)</span></br>
 +
<span id="little">Bxb1 GTG cond. #1 - #2: 79.0ng/uL (purity: 1.89)</span></br>
 +
<span id="little">Bxb1 GTG cond. #2 - #1: 110.7ng/uL (purity: 1.89)</span></br>
 +
<span id="little">Bxb1 GTG cond. #2 - #2: 66.7ng/uL (purity: 1.94)</span></br>
 +
 +
</br></br>
 +
<b>Discussions</b></br></br>
 +
<span id="little"> Bxb1 GTG cond. #1 - #1 and Bxb1 GTG cond. #2 - #1 will be used to cloning.</span></br>
 +
</div>
 +
</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
 +
 +
<div class="note-title">Insert DNA (Bxb1 GTG) and vector (pTrc and pBAD) cloning</div>
 +
 +
<div id="content_note" >
 +
 +
<span id="little">Insert DNA: Bxb1 integrase mutated by primer.</span></br>
 +
<span id="little">Vectro DNA: pTrcHis2A, pBAD-mycHisC</span></br>
 +
<span id="little">Prepared DNA concentration:</span></br>
 +
<span>
 +
<ul style='list-style-type:square'>
 +
<li>Bxb1 GTG cond. #1 - #1: 128.2ng/uL (purity: 1.89)</li>
 +
<li>Bxb1 GTG cond. #2 - #1: 110.7ng/uL (purity: 1.89)</li>
 +
<li>pTrcHis2A: 116.1ng/uL</li>
 +
<li>pBAD-mycHisC: 128.7ng/uL</li>
 +
</ul>
 +
</span>
 +
</br></br>
 +
<span id="little">Enzyme digestion: 37℃ for 1hr 30min, 80℃ for 20min</span></br></br>
 +
 +
<div align="center"><img id="figure" alt="2_0827Fig2" src="http://2012.igem.org/wiki/images/c/c7/KAIST_2nd_08272012_Fig2.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br></br>
 +
 +
<span id="little">Dephosphorylation: 37℃ for 30min, 70℃ for 5min</span></br></br>
 +
<div align="center"><img id="figure" alt="2_0827Fig3" src="http://2012.igem.org/wiki/images/6/67/KAIST_2nd_08272012_Fig3.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br></br>
 +
<span id="little">Ligation: 16℃ overnight</span></br></br>
 +
<div align="center"><img id="figure" alt="2_0827Fig4" src="http://2012.igem.org/wiki/images/8/8b/KAIST_2nd_08272012_Fig4.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
</div>
 +
</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
 +
 +
<div class="note-title">Bxb1, pPoC double transformants colony PCR</div>
 +
 +
<div id="content_note" >
 +
 +
<span id="little">Colony #1, 4, 7, 10, 13, 14, 16, 19 and 22 from master plate</span>
 +
</br></br>
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="2_0827Fig5" src="http://2012.igem.org/wiki/images/4/45/KAIST_2nd_08272012_Fig5.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br></br>
 +
<b>Discussions</b></br></br>
 +
 +
<span id="little">The gel showed that all the colonies have Bxb1-pTrc vector. And also they all have pPoC or pPoCpi, because they show color.</span>
 +
 +
</br>
 +
</div>
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
 +
</section>
</section>
<section id="28">
<section id="28">
<div class="date">August 28<sup>th</sup> 2012</div></br>
<div class="date">August 28<sup>th</sup> 2012</div></br>
 +
<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='http://2012.igem.org/wiki/images/2/22/KAIST_FlipFlop.png'/> Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></div></br>
 +
 +
<div class="note-title">Bxb1-GTG-pTrc and Bxb1-GTG-pBAD electrotransformation into MG1655</div>
 +
 +
<div id="content_note" >
 +
 +
</div>
 +
 +
</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
 +
 +
<div class="note-title">Bxb1-pTrc and Bxb1-pBAD transformants check</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Results</b></br></br>
 +
 +
<span>Bxb1-pTrc: No colony</br>
 +
Bxb1-pBAD: Colonies appeared and incoculated into 3mL of LB containing 1% of Ap</br>
 +
</span>
 +
 +
</br>
 +
</div>
 +
</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
 +
 +
<div class="note-title">Bxb1-pTrc and Bxb1-pBAD transformants colony PCR</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Results</b></br></br>
 +
<div align="center"><img id="figure" alt="2_0828Fig1" src="http://2012.igem.org/wiki/images/1/15/KAIST_2nd_08282012_Fig1.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br></br>
 +
 +
</br>
 +
</div>
 +
</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
 +
 +
<div class="note-title">Bxb1-pTrc and Bxb1-pBAD transformants plasmid DNA miniprep</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Results</b></br></br>
 +
<span>Colony #2: </br>
 +
Colony #3:</br>
 +
</span>
 +
</br></br>
 +
</div>
 +
</br><div align="center"><img style="width:670px; height:2px;" src="http://2012.igem.org/wiki/images/7/7f/Horizontal_line_kaist.png"></img></div></br>
 +
 +
<div class="note-title">mRFP, GFP 30mL culture and sampling</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Results</b></br></br>
 +
 +
</div>
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
</section>
</section>
 +
<section id="29">
<section id="29">
<div class="date">August 29<sup>th</sup> 2012</div></br>
<div class="date">August 29<sup>th</sup> 2012</div></br>
 +
<div id="content_note" >
 +
<span id="little">No Special Event!</span>
 +
</br>
 +
 +
</div>
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
</section>
</section>
<section id="30">
<section id="30">
<div class="date">August 30<sup>th</sup> 2012</div></br>
<div class="date">August 30<sup>th</sup> 2012</div></br>
 +
<div id="content_note" >
 +
<span id="little">No Special Event!</span>
 +
</br>
 +
 +
</div>
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
</section>
</section>
<section id="31">
<section id="31">
<div class="date">August 31<sup>st</sup> 2012</div></br>
<div class="date">August 31<sup>st</sup> 2012</div></br>
-
</section>
+
<div id="content_note" >
-
 
+
<span id="little">No Special Event!</span>
 +
</br>
 +
 +
</div>
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 +
</section>
</br></br>
</br></br>
</div>
</div>
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Revision as of 06:52, 23 September 2012

KAIST Korea 2012 iGEM

Notebook : Labnote-August

Labnote

August

August 1st 2012

 PACKMAN

PCR amplification of Moth_1191, 1199 gene : decreasing annealing temperature down to 45℃
To make sure there’s no problem with unspecific binding or annealing temperature, we decreased annealing condition down to 45℃.

Results

0801Fig1

There was no band and it became certain that there are no problems with annealing procedure.


Induction of Moth_0109 gene (sampling) and running SDS-PAGE gel.
We induced Moth_0109 gene in pBAD/myc vector with different Arabinose conditions and temperature: 10mM, 30mM and 37℃, 30℃. And then, we run the sample into SDS-PAGE gel. The gel is stained with coomassie blue for 1 hour and de-stained for overnight.



PCR amplification of Moth_1191, 1199 gene with Gibson and TOPO primer
To see whether there are any problem with Gibson primers that we’ve designed, we did one more pcr amplification. Primers for Gibson assembly and TOPO cloning are mixed, and we could see any problems with forward or reverse primers.

Results

0801Fig2

Each lane indicates:

  • Moth_1191_1 : TOPO forward primer and Gibson reverse primer
  • Moth_1191_2 : Gibson forward primer and TOPO reverse primer
  • Moth_1199_1 : Gibson forward primer and TOPO reverse primer
  • Moth_1199_2 : TOPO forward primer and Gibson reverse primer

Correct size of band appeared when topo forward primer and Gibson reverse primer was used in amplification of both genes. Therefore, we can conclude that there is some unknown problem with forward primers.

Back to the Calendar
August 2nd 2012

 PACKMAN

Primer design for FDH derived from Candida boidinii mutant
Results

0802Fig1

Discussion

These primers are universial primer that can be used for insertion of this gene into both pTrcHis2A vector and pBAD/mycHisC vector.


Expression check of Moth_0109 gene on pBAD/mycHisC vector (Result)
For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 59.94 kDa protein, formate tetrahydrofolate ligase.

Results

0802Fig2

Discussion

The band of PAGE gel was not certain, so we
Back to the Calendar
August 3rd 2012

No Special Event!
Back to the Calendar
August 4th 2012

 PACKMAN

1191 and 1199 forward primer design
We designed new forward primers for both 1191 and 1199 gene that are thought to solve problems appeared during PCR amplification.

Results

0804Fig1

Back to the Calendar
August 5th 2012

No Special Event!
Back to the Calendar
August 6th 2012

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

 PACKMAN

FDH gene PCR amplification
Results

0807Fig1

Discussion

We amplified 2 samples of formate dehydrogenase gene with the primers we designed. From gel electrophoresis picture, we can say that oligonucleotides with the proper size 1100bp are present in the sample.
0807Fig2


Back to the Calendar
August 8th 2012

 PACKMAN

FDH gene and pTrcHis2A, pBAD/mycHisC vector enzyme digestion
Procedure

We mixed restriction enzymes and buffers with DNA samples as shown below.

0808Fig1

After mixing the enzymes and buffer with the sample, we incubated the sample on 37℃ for 1 hour and then on 65℃ for 20minutes.


pTrcHis2A, pBAD/myc vector dephosphorylation
Procedure

We mixed phosphatidase and buffer with DNA sample as shown below.

0808Fig2

After mixing the enzyme and buffer with the sample, we incubated the sample on 37℃ for 1 hour 20minutes and then on 70℃ for 5minutes.


ligation of FDH gene with pTrcHis2A and pBAD/mycHisC vectors
Procedure

We mixed ligase and buffer with DNA sample as shown below.

0808Fig3

After mixing the enzyme and buffer with the sample, we incubated the sample on 16℃ for 16 hours.


Primer amplification of 1191 and 1199
Results

0808Fig4

Discussion

These
Back to the Calendar
August 9th 2012

 PACKMAN

vector transformation into TOP10 cell
Transformation of pTrcHis2A vector into TOP10 cell was successful. So we did vector mini-preparation after the transformation. However, transformation of pBAD vector has failed, so we ligated the samples again.

Results

0809Fig1

This is gel picture to confirm that FDH is well inserted into TOP10 cells. 3 wells on the left are PCR product of
Back to the Calendar
August 10th 2012

No Special Event!
Back to the Calendar
August 11th 2012

 PACKMAN

pTrcHis2A vector transformation into E.coli MG1655 cell
Procedure



pBAD/mycHisC-FDH transformation into TOP10

Back to the Calendar
August 12th 2012

 PACKMAN

colony PCR check of pTrcHis2A inserted FDH gene
Results

0812Fig1

Discussion

We checked the presence of FDH gene by conducting colony PCR. Correct size of band appeared at about 1115bp, which means formate dehydrogenase is inserted correctly.


pBAD FDH MG1655 electroporation
Results

Fail
Back to the Calendar
August 13th 2012

 PACKMAN

FDH pBAD MG1655 electroporation
Procedure

We seeded cells on 7:10 AM and did cell-down on 10:00AM. And then we plated cells on 1:00PM and checked colony on 8/14 5:00AM

Results




FDH pTrc MG1655 expression check (sampling and induction)
Procedure

We seeded cells (500λ of cells put into 50mL LB with 50λ Ampicillin) on 7 : 30 AM and checked Optical Density on 10:30pm. According to the O.D. value, we induced 37℃ sample on 10:30PM and did sampling on 4:30pm. Also, we induced 30℃ sample on 11:30pm and did sampling on 5:30pm. (6 hour induction) Each temperature samples were induced by different conditions ; 2mM and 3mM of IPTG.

Back to the Calendar
August 14th 2012

 PACKMAN

colony PCR check of pBADmycHisC inserted FDH gene
Results

0814Fig1

Discussion

We checked the presence of FDH gene by conducting colony PCR. Correct size of band appeared at about 1115 bp, which means formate dehydrogenase is inserted correctly.


Expression check of Formate dehydrogenase on pTrcHis2A vector (Result)
Results

0814Fig2

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 43.7 kDa protein, formate dehydrogenase. The band appeared significantly on the correct size.


FDH pBAD MG1655 seeding for expression check
Procedure

We seeded cell on 7:00PM, so we’ll incubate cell about 16hours. (This means we have to do next seeding on 11:00AM. )


Primer design for the insertion of 1202 and 0109
Results

0814Fig3

Discussion

To eliminate uncertain spacer between genes, we inserted
Back to the Calendar
August 15th 2012

 PACKMAN

FDH pBAD MG1655 expression check
Procedure

We seeded cells on 11 : 30 AM and checked Optical Density on 1:30pm. According to the O.D. value, we induced 37℃ sample on 1:40pm and did sampling on 7:40pm. Also, we induced 30℃ sample on 3:30pm and did sampling on 9:30pm. (6 hour induction) Each temperature samples were induced by different conditions: 10mM of arabinose and 30mM of arabinose.
Back to the Calendar
August 16th 2012

 PACKMAN

PCR amplification of 1201, 1202, 1203, 1198 and 1191
Results

0816Fig1

Discussion

Primers arrived, and we began to do additional PCR amplification on the preparation for Gibson assembly. Each PCR sample has the correct size of oligonucleotides except Moth 1202 gene.

  • 1191 : 921bp
  • 1198 : 972bp + about 400bp = 1300 bp
  • 1201 : 1341bp + ?
  • 1202 : 2190bp +
  • 1203 : 2025bp + 1600bp = 3625bp

We did PCR purification on the PCR product and the concentration/purity result was as shown below.


0816Fig2



Pre-culture of 0109
Discussion

There was no vector prepared for Moth 0109, so we did 5ml pre-culture on 10ml tube. Cell was grown about 16hours after pre-culture, and we did vector mini-prep on the sample. The concentration of the mini-prepped vector is 726.8ng/ul and the purity(A260/A280) was 1.86.

 Flip Flop

pPoC and pPoCpi electro-transformation into MG1655
Strain: MG1655

Plasmid source:

  • pPoC: from DH5α-pPoC colony #1 and TOP10-pPoC colony #1
  • pPoCpi: from TOP10-pPoC colony #1 and #3


Back to the Calendar
August 17th 2012

 PACKMAN

Expression check of Formate dehydrogenase.
Results

0817Fig1

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 40.xx kDa protein, formate dehydrogenase. The band appeared more significantly than the case of pTrcHis2A vector. Therefore, using pBAD promoter seems to produce more proteins.


PCR amplification – second trial
Results

0817Fig2

Discussion

As the band was not significant for 1203 and 1198 on previous trial and purity was low for 1191, we tried secondary PCR amplification on samples for Gibson assembly; 1202, 1203, 1198, 1191. Particularly for 1202 gene, we tried additional colony PCR, so that we could check any problem with vector mini-prep. (PCR solution for 1202 colony PCR was : 22ul

0817Fig3


 Flip Flop

MG1655-pPoC, MG1655-pPoCpi streaking
We could not distinguish single colony from plates. Therefore we scraped cells and streaked onto new plates.

Discussions

Wild-type E.coli showed more reddish and yellowish color than competent strains, DH5α and TOP10. The promoter BBa_J23119 may show different strength in-between strains.


TOP10-pTrcHis2A_Bxb1 expression check
TOP10 containing pTrcHis2A was cultured in 30mL of LB with 1% of Ampicillin.
IPTG induced when OD = 0.4
Sampling: 8 hours after induction, 1010 cells.
Only soluble fractions were PAGEed.

Back to the Calendar
August 18th 2012

 PACKMAN

PCR amplification of 1202, 0109 gene with original primer and Gibson primers
Results

0818Fig1

Each lane indicates:

  • Moth_1202_1 : Gibson forward + original reverse
  • Moth_1202_2 : Gibson reverse + original forward
  • Moth_0109_1 : Gibson forward + original reverse
  • Moth_0109_2 : Gibson reverse + original forward

There was correct size and unspecific size of band on 1202_1 sample. Although there are also unspecific band, 1202_2 sample has large band of correct size. 0109_1 sample has only unspecific band and 0109_2 sample has right size of band.


Discussion

When the PCR was unsuccessful, we first thought it might be problem of forward primer as in the case of 1199 and 1191. Our guess was correct, and we’re going to fix the problem with forward primers.


1202 and 0109 forward primer design
We designed new forward primer hoping no problem with it.

Results


 Flip Flop

MG1655-pPoC, MG1655-pPoCpi colony inoculation
Two colonies from each plate were inoculated into 3mL of LB containing 1% of chloramphenicol.

Discussions

In 12 hours, cell cultures show colors compared to another common E.coli culture. When they are centrifuged, pellet shows clear and definite yellow or red color.


pTrcHis2A_Bxb1 electro-transformation into MG1655-pPoC, MG1655-pPoCpi
pTrcHis2A_Bxb1 is electro-transformed into MG1655 containing pPoC and pPoCpi.

Plasmid source: TOP10-pTrcHis2A_Bxb1


Back to the Calendar
August 19th 2012

No Special Event!
Back to the Calendar
August 20th 2012

No Special Event!
Back to the Calendar
August 21st 2012

No Special Event!
Back to the Calendar
August 22nd 2012

 Flip Flop

Primer design for Bxb1 integrase cloning
Primers for Bxb1 integrase cloning were designed. For controlling basal transcription level optimization, a mutant primer which has GTG rather than ATG for its start codon was also ordered.

Results

2_0822Fig1

Back to the Calendar
August 23rd 2012

No Special Event!
Back to the Calendar
August 24th 2012

No Special Event!
Back to the Calendar
August 25th 2012

 Flip Flop

pPoC-Bxb1 double transformants inoculation for prep & PCR check
Double transformants 1 through 24 were inoculated into 3ml of LB containing 1% of Cm and Ap. For our convenience, three colonies were inoculated in one culture tube.

Back to the Calendar
August 26th 2012


 Flip Flop

pPoC-Bxb1 double transformants plasmid DNA miniprep & PCR check
Inoculated cultures were mipi-preped. And Bxb1-pTrc plasmid was checked by PCR. In case of pPoC, it would be check by color.

Results

Vector

2_0826Fig1

Confirm PCR

2_0826Fig2


Discussions

In confirm PCR, all the colonies seem to have Bxb1 vector. Colony #1, 4, 7, 10, 13, 14, 16, 19 and 22 will be inoculated.


Bxb1 PCR with new primers
For cloning of Bxb1 into pBAD and mutating its start codon from ATG to GTG, we PCR pGEM-B1-Bxb1 vector as template. Samples were duplicated

PCR conditions
  1. 95℃ for 3min
  2. 95℃ for 30sec
  3. 55℃ for 30sec
  4. 72℃ for 1min 30sec
  5. Step 2 to 4: 34 cycles
  6. 72℃ for 10min
  7. 12℃ forever


Results

2_0826Fig3

Duplicated samples were purified in one DNA binding column.
ATG: 297.9ng/uL (purity: 1.90)
GTG: purify failed. Sample lost

Discussions

There were multiple bands in our PCR products. We will PCR again Bxb1-GTG with different conditions. Template vector may show larger bands.


pGEM-Bxb1 template vector gel electrophoresis
To check whether the undesired bands were template vector, we diluted template vector with same conditions of PCR, 50X, and electrophoresized.

Results

2_0826Fig4


Discussion

The band was different from undesired bands from Bxb1 ATG, GTG PCR products.


Insert DNA (Bxb1 integrase) and vector (pTrc and pBAD) cloning
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August 27th 2012


 Flip Flop

Bxb1-pTrc and Bxb1-pBAD electrotransformation into MG1655


Bxb1 PCR with changed conditions
For cloning of Bxb1 into pBAD and mutating its start codon from ATG to GTG, we PCR pGEM-B1-Bxb1 vector as template. Samples were duplicated

PCR conditions #1 PCR conditions #2
1) 95℃ for 3min 1) 95℃ for 3min
2) 95℃ for 30sec 2) 95℃ for 30sec
3) 56.5℃ for 30sec 3) 58℃ for 30sec
4) 72℃ for 1min 30sec
  (Step 2 to 4: 34 cycles)
4) 72℃ for 1min 30sec
  (Step 2 to 4: 34 cycles)
5) 72℃ for 10min 5) 72℃ for 10min
6) 12℃ forever 6) 12℃ forever
Results

2_0827Fig1


Discussions

Still, undesired bands appeared. We do not know where they come from. We decide to extract desired DNA with gel extraction.


Bxb1, with GTG as start codon, PCR product gel extraction
PCR products were gel extracted.

Results

Bxb1 GTG cond. #1 - #1: 128.2ng/uL (purity: 1.89)
Bxb1 GTG cond. #1 - #2: 79.0ng/uL (purity: 1.89)
Bxb1 GTG cond. #2 - #1: 110.7ng/uL (purity: 1.89)
Bxb1 GTG cond. #2 - #2: 66.7ng/uL (purity: 1.94)


Discussions

Bxb1 GTG cond. #1 - #1 and Bxb1 GTG cond. #2 - #1 will be used to cloning.


Insert DNA (Bxb1 GTG) and vector (pTrc and pBAD) cloning
Insert DNA: Bxb1 integrase mutated by primer.
Vectro DNA: pTrcHis2A, pBAD-mycHisC
Prepared DNA concentration:
  • Bxb1 GTG cond. #1 - #1: 128.2ng/uL (purity: 1.89)
  • Bxb1 GTG cond. #2 - #1: 110.7ng/uL (purity: 1.89)
  • pTrcHis2A: 116.1ng/uL
  • pBAD-mycHisC: 128.7ng/uL


Enzyme digestion: 37℃ for 1hr 30min, 80℃ for 20min

2_0827Fig2


Dephosphorylation: 37℃ for 30min, 70℃ for 5min

2_0827Fig3


Ligation: 16℃ overnight

2_0827Fig4



Bxb1, pPoC double transformants colony PCR
Colony #1, 4, 7, 10, 13, 14, 16, 19 and 22 from master plate

Results

2_0827Fig5


Discussions

The gel showed that all the colonies have Bxb1-pTrc vector. And also they all have pPoC or pPoCpi, because they show color.
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August 28th 2012

 Flip Flop

Bxb1-GTG-pTrc and Bxb1-GTG-pBAD electrotransformation into MG1655


Bxb1-pTrc and Bxb1-pBAD transformants check
Results

Bxb1-pTrc: No colony
Bxb1-pBAD: Colonies appeared and incoculated into 3mL of LB containing 1% of Ap



Bxb1-pTrc and Bxb1-pBAD transformants colony PCR
Results

2_0828Fig1





Bxb1-pTrc and Bxb1-pBAD transformants plasmid DNA miniprep
Results

Colony #2:
Colony #3:




mRFP, GFP 30mL culture and sampling
Results

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August 29th 2012

No Special Event!
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August 30th 2012

No Special Event!
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August 31st 2012

No Special Event!
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