Team:KAIST Korea/Notebook Labnote/2012 9
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<b>Procedure</b></br></br> | <b>Procedure</b></br></br> | ||
- | <span id="little">We seeded cells (500λ of cells put into 50mL LB with 50λ Ampicillin) and checked Optical Density. According to the O.D. value, we induced 37℃ and 30℃ sample and did sampling. Each samples were induced by different conditions ; 1mM and 2mM of IPTG, 10mM and 30mM of arabinose. | + | <span id="little">We seeded cells (500λ of cells put into 50mL LB with 50λ Ampicillin) and checked Optical Density. According to the O.D. value, we induced 37℃ and 30℃ sample and did sampling. Each samples were induced by different conditions ; 1mM and 2mM of IPTG, 10mM and 30mM of arabinose.</br></br></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="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> | <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> | ||
</section> | </section> | ||
<section id="9"> | <section id="9"> | ||
<div class="date">September 9<sup>th</sup> 2012</div></br> | <div class="date">September 9<sup>th</sup> 2012</div></br> | ||
- | <div id="content_note" > | + | <img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></br> |
- | <span id="little"> | + | <div class="note-title">Moth_0109 expression optimization _ gel picture</div> |
- | </br> | + | |
+ | <div id="content_note" > | ||
+ | |||
+ | <b>Results</b></br></br> | ||
+ | |||
+ | <div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/6/64/120909.PNG"></img></div> | ||
+ | <div style="clear:both;"></div> | ||
+ | |||
+ | <b>Discussion</b></br></br> | ||
+ | <span id="little">For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 59.94kDa protein, formate--tetrahydrofolate ligase. The gel was ruined during staining/destaining procedure, and we couldn’t find any band for this protein. </br> | ||
+ | Therefore, we concluded that expression of 0109 gene on pBAD vector has failed and decided to try once more.</br></br></span> | ||
+ | </div> | ||
+ | </br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br> | ||
+ | |||
+ | <div class="note-title">Moth_1191 expression optimization _ gel picture </div> | ||
+ | |||
+ | <div id="content_note" > | ||
+ | |||
+ | <b>Results</b></br></br> | ||
+ | <div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/e/e0/120909_-_1.PNG"></img></div> | ||
+ | <div style="clear:both;"></div> | ||
+ | |||
+ | </br></br> | ||
+ | |||
+ | <b>Discussion</b></br></br> | ||
+ | |||
+ | <span id="little">For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 33.92kDa protein, methylenetetrahydrofolate reductase. The gel was ruined during staining/destaining procedure, and we couldn’t find any band for this protein. </br> | ||
+ | Therefore, we concluded that expression of 1191 gene on pBAD vector has failed and decided to try once more. | ||
+ | </br></br></span> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | </br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br> | ||
+ | |||
+ | <div class="note-title">FDH pTrc expression optimization _ gel picture </div> | ||
+ | |||
+ | <div id="content_note" > | ||
+ | |||
+ | <b>Results</b></br></br> | ||
+ | |||
+ | <div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/b/bf/120909-2.PNG"></img></div> | ||
+ | <div style="clear:both;"></div> | ||
+ | |||
+ | </br></br> | ||
+ | |||
+ | <b>Discussion</b></br></br> | ||
+ | |||
+ | <span id="little">For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 40.37kDa protein, Formate dehydrogenase. The gel was ruined during staining/destaining procedure, and we couldn’t find any band for this protein. </br> | ||
+ | Therefore, we concluded that expression of FDH gene on pTrc vector has failed and decided to try once more. | ||
+ | </br></br></span> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | </br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br> | ||
+ | |||
+ | <div class="note-title">FDH pBAD expression optimization _ gel picture</div> | ||
+ | |||
+ | <div id="content_note" > | ||
+ | |||
+ | <b>Results</b></br></br> | ||
+ | |||
+ | <div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/6/6d/120909-3.PNG"></img></div> | ||
+ | <div style="clear:both;"></div> | ||
+ | |||
+ | </br></br> | ||
+ | |||
+ | <b>Discussion</b></br></br> | ||
+ | |||
+ | <span id="little">For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 40.37kDa protein, Formate dehydrogenase. The gel was ruined during staining/destaining procedure, and we couldn’t find any band for this protein. </br> | ||
+ | Therefore, we concluded that expression of FDH gene on pTrc vector has failed and decided to try once more. | ||
+ | </br></br></span> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | |||
+ | </br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br> | ||
+ | |||
+ | <div class="note-title">Primer design to PCR-amplify piBR181 vector.</div> | ||
+ | |||
+ | <div id="content_note" > | ||
+ | |||
+ | <b>Results</b></br></br> | ||
+ | |||
+ | <div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/d/d5/1209094.PNG"></img></div> | ||
+ | <div style="clear:both;"></div> | ||
+ | |||
+ | </br></br> | ||
+ | |||
+ | <b>Discussion</b></br></br> | ||
+ | |||
+ | <span id="little">For the Gibson assembly with RE digestion of vector, we had several problems with de-phosphorylation and purification after de-phosphorylation. Therefore we decided to PCR-amplify the piBR181 vector and use the PCR product in Gibson assembly. </br></br></span> | ||
+ | |||
</div> | </div> | ||
Line 450: | Line 541: | ||
<section id="10"> | <section id="10"> | ||
<div class="date">September 10<sup>th</sup> 2012</div></br> | <div class="date">September 10<sup>th</sup> 2012</div></br> | ||
+ | |||
+ | <img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></br> | ||
+ | <div class="note-title">PCR amplification of 1202 gene with new primers</div> | ||
+ | <div id="content_note" > | ||
+ | |||
+ | <b>Results</b></br></br> | ||
+ | |||
+ | <div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/a/ad/120910.PNG"></img></div> | ||
+ | <div style="clear:both;"></div> | ||
+ | |||
+ | <b>Discussion</b></br></br> | ||
+ | <span id="little">The correct size of band appeared for the second PCR product, but there was no band appeared for the first PCR product. </br></br></span> | ||
+ | </div> | ||
<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="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/> Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></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="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/> Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br> | ||
Line 480: | Line 584: | ||
<section id="11"> | <section id="11"> | ||
<div class="date">September 11<sup>th</sup> 2012</div></br> | <div class="date">September 11<sup>th</sup> 2012</div></br> | ||
- | |||
- | |||
- | |||
+ | <img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></br> | ||
+ | <div class="note-title">PCR amplification of piBR181 vector</div> | ||
+ | |||
+ | <div id="content_note" > | ||
+ | |||
+ | <b>Results</b></br></br> | ||
+ | |||
+ | <div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/6/66/120911.PNG"></img></div> | ||
+ | <div style="clear:both;"></div> | ||
+ | |||
+ | <b>Discussion</b></br></br> | ||
+ | <span id="little">The correct size of piBR181 vector pcr product is 5297bp. There appeared the right size and unspecific band on the gel. So we decided to do pcr once more.</br></br></span> | ||
</div> | </div> | ||
+ | |||
+ | </br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br> | ||
+ | |||
+ | <div class="note-title">Pre-culture of 0109, 1191, FDH containing cells.</div> | ||
+ | |||
+ | <div id="content_note" > | ||
+ | |||
+ | <b>Procedure</b></br></br> | ||
+ | |||
+ | <span id="little">We did 5ml pre-culture on 10ml tube for the cells containing each gene. Cell was grown about 16hours after pre-culture, and then induced with proper inducer.</br></br></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="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> | <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> | ||
</section> | </section> | ||
<section id="12"> | <section id="12"> | ||
- | + | <div class="date">September 12<sup>th</sup> 2012</div></br> | |
+ | |||
+ | <img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></br> | ||
+ | <div class="note-title">PCR amplification of piBR181 vector : 10 sample</div> | ||
+ | |||
+ | <div id="content_note" > | ||
+ | |||
+ | <b>Results</b></br></br> | ||
+ | |||
+ | <div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/6/65/120912.PNG"></img></div> | ||
+ | <div style="clear:both;"></div> | ||
+ | |||
+ | <b>Discussion</b></br></br> | ||
+ | <span id="little">The full PCR construct of piBR181 vector is 5297bp. From the gel image, we could find that right size of piBR181 PCR product clearly exists. </br></br></span> | ||
+ | </div> | ||
+ | |||
+ | </br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br> | ||
+ | |||
+ | <div class="note-title">FDH pTrc & pBAD/0109 pBAD/1191 pBAD MG1655 expression check (sampling and induction)</div> | ||
+ | |||
+ | <div id="content_note" > | ||
+ | |||
+ | <b>Procedure</b></br></br> | ||
+ | |||
+ | <span id="little">We seeded cells (500λ of cells put into 50mL LB with 50λ Ampicillin) and checked Optical Density. According to the O.D. value, we induced 37℃ and 30℃ sample and did sampling. Each samples were induced by different conditions ; 1mM and 2mM of IPTG, 10mM and 30mM of arabinose.</br></br></span> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | |||
+ | |||
<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="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/> Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></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="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/> Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br> | ||
Line 518: | Line 674: | ||
<section id="13"> | <section id="13"> | ||
<div class="date">September 13<sup>th</sup> 2012</div></br> | <div class="date">September 13<sup>th</sup> 2012</div></br> | ||
+ | <img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></br> | ||
+ | <div class="note-title">Moth_0109 pBAD expression check</div> | ||
+ | |||
<div id="content_note" > | <div id="content_note" > | ||
- | |||
- | |||
+ | <b>Results</b></br></br> | ||
+ | |||
+ | <div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/d/da/120913_-_1.PNG"></img></div> | ||
+ | <div style="clear:both;"></div> | ||
+ | |||
+ | <b>Discussion</b></br></br> | ||
+ | <span id="little">For each samples of different induction conditions, we ran 10% SDS-PAGE gel and checked the presence of 59.94kDa protein, formate--tetrahydrofolate ligase. We couldn’t find any band for this protein. </br> | ||
+ | There was no significant band on the gel, so we couldn’t conclude that this protein is expressed. Our opinion is that we should run Western blot to exactly find this protein. </br></br></span> | ||
+ | </div> | ||
+ | </br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br> | ||
+ | |||
+ | <div class="note-title">Moth_1191 pBAD expression check</div> | ||
+ | |||
+ | <div id="content_note" > | ||
+ | |||
+ | <b>Results</b></br></br> | ||
+ | <div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/a/af/120913-2.PNG"></img></div> | ||
+ | <div style="clear:both;"></div> | ||
+ | |||
+ | </br></br> | ||
+ | |||
+ | <b>Discussion</b></br></br> | ||
+ | |||
+ | <span id="little">For each samples of different induction conditions, we run 15% SDS-PAGE gel and checked the presence of 33.92kDa protein, methylenetetrahydrofolate reductase. Although we were not able to discriminate each band of insoluble fraction, We could find that there exists right band at the right size. | ||
+ | </br></br></span> | ||
+ | |||
</div> | </div> | ||
+ | |||
+ | </br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br> | ||
+ | |||
+ | <div class="note-title">FDH pBAD expression check</div> | ||
+ | |||
+ | <div id="content_note" > | ||
+ | |||
+ | <b>Results</b></br></br> | ||
+ | |||
+ | <div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/9/9e/120913-3.PNG"></img></div> | ||
+ | <div style="clear:both;"></div> | ||
+ | |||
+ | </br></br> | ||
+ | |||
+ | <b>Discussion</b></br></br> | ||
+ | |||
+ | <span id="little">For each samples of different induction conditions, we run 8% SDS-PAGE gel and checked the presence of 40.37 kDa protein, Formate dehydrogenase. We couldn’t find any band for this protein. </br> | ||
+ | There was no significant band on the gel, so we couldn’t conclude that this protein is expressed. Our opinion is that we should run Western blot to exactly find this protein. | ||
+ | </br></br></span> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | </br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br> | ||
+ | |||
+ | <div class="note-title">FDH pTrc expression check</div> | ||
+ | |||
+ | <div id="content_note" > | ||
+ | |||
+ | <b>Results</b></br></br> | ||
+ | |||
+ | <div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/1/1b/120913-4.PNG"></img></div> | ||
+ | <div style="clear:both;"></div> | ||
+ | |||
+ | </br></br> | ||
+ | |||
+ | <b>Discussion</b></br></br> | ||
+ | |||
+ | <span id="little">For each samples of different induction conditions, we run 8% SDS-PAGE gel and checked the presence of 40.37 kDa protein, Formate dehydrogenase. We couldn’t find any band for this protein. </br> | ||
+ | There was no significant band on the gel, so we couldn’t conclude that this protein is expressed. Our opinion is that we should run Western blot to exactly find this protein. | ||
+ | </br></br></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="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> | <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> | ||
</section> | </section> | ||
<section id="14"> | <section id="14"> | ||
<div class="date">September 14<sup>th</sup> 2012</div></br> | <div class="date">September 14<sup>th</sup> 2012</div></br> | ||
- | < | + | <img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></br> |
- | + | <div class="note-title">Gibson assembly – assembly of pcr fragments (pcr fragment of vector added)</div> | |
- | + | ||
+ | <div id="content_note" > | ||
- | + | <b>Results</b></br></br> | |
+ | <span id="little">Among three methods to assemble DNA fragments, we chose third method, which is assembly of PCR amplified DNA fragments and vector. | ||
+ | 0.17umoles of each fragments are added into the reaction sample, and volume to be added for each fragments has calculated on the assumption that average MW of each base is 660Da. Table below is the result of calculation.</br></br></span> | ||
+ | |||
+ | |||
+ | <div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/b/b7/120914_f1.PNG"></img></div> | ||
+ | <div style="clear:both;"></div> | ||
+ | |||
+ | <div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/8/8b/120914_f2.PNG"></img></div> | ||
+ | <div style="clear:both;"></div> | ||
+ | |||
+ | <span id="little"></br>After 1 hour of incubation on 50℃, the vector was transformed into TOP10 and NEB competent cell. Cells were plated and grown overnight during colony formation. </br></br></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="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> | <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> | ||
</section> | </section> | ||
Line 554: | Line 794: | ||
<section id="16"> | <section id="16"> | ||
<div class="date">September 16<sup>th</sup> 2012</div></br> | <div class="date">September 16<sup>th</sup> 2012</div></br> | ||
+ | |||
+ | <img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></br> | ||
+ | <div class="note-title">Gibson assembly colony pcr</div> | ||
+ | |||
+ | <div id="content_note" > | ||
+ | |||
+ | <b>Results</b></br></br> | ||
+ | |||
+ | <div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/f/fb/120916.PNG"></img></div> | ||
+ | <div style="clear:both;"></div> | ||
+ | |||
+ | <b>Discussion</b></br></br> | ||
+ | <span id="little">As we couldn’t get full fragment by colony-pcr, we did 3ml culture, plasmid mini-prep and PCR for each colony. This will take one more day to get result.</br></br></span> | ||
+ | </div> | ||
<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="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/> Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></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="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/> Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br> | ||
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<section id="17"> | <section id="17"> | ||
<div class="date">September 17<sup>th</sup> 2012</div></br> | <div class="date">September 17<sup>th</sup> 2012</div></br> | ||
+ | |||
+ | <img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></br> | ||
+ | <div class="note-title">Gibson assembly fragment PCR after prep</div> | ||
+ | |||
+ | <div id="content_note" > | ||
+ | |||
+ | <b>Results</b></br></br> | ||
+ | |||
+ | <div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/f/f2/120917.PNG"></img></div> | ||
+ | <div style="clear:both;"></div> | ||
+ | |||
+ | <b>Discussion</b></br></br> | ||
+ | <span id="little">There appeared correct size of band with several unspecific bands on the gel. We couldn’t even try to do gel extraction because the band was too thin and faint. We decided to do more rounds of pcr amplification.</br></br></span> | ||
+ | </div> | ||
<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="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/> Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></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="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/> Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br> | ||
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<section id="19"> | <section id="19"> | ||
<div class="date">September 19<sup>th</sup> 2012</div></br> | <div class="date">September 19<sup>th</sup> 2012</div></br> | ||
+ | |||
+ | <img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></br> | ||
+ | <div class="note-title">Gibson F1 PCR after prep</div> | ||
+ | |||
+ | <div id="content_note" > | ||
+ | |||
+ | <b>Results</b></br></br> | ||
+ | |||
+ | <div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/e/eb/120919-1.PNG"></img></div> | ||
+ | <div style="clear:both;"></div> | ||
+ | |||
+ | <b>Discussion</b></br></br> | ||
+ | <span id="little">We did PCR amplification after prep of F1 vector, but there appeared no band on the gel. </br></br></span> | ||
+ | </div> | ||
+ | </br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br> | ||
+ | <div class="note-title">Gibson F2 5-1~5, F1-3-1~5, F1-4-1~5</div> | ||
+ | |||
+ | <div id="content_note" > | ||
+ | |||
+ | <b>Results</b></br></br> | ||
+ | |||
+ | <div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/3/3c/120919-2.PNG"></img></div> | ||
+ | <div style="clear:both;"></div> | ||
+ | |||
+ | <b>Discussion</b></br></br> | ||
+ | <span id="little">There appeared several unspecific bands on the gel and </br></br></span> | ||
+ | </div> | ||
+ | |||
<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="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/> Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></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="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/> Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br> | ||
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<section id="27"> | <section id="27"> | ||
<div class="date">September 27<sup>th</sup> 2012</div></br> | <div class="date">September 27<sup>th</sup> 2012</div></br> | ||
- | < | + | <img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></br> |
- | + | <div class="note-title">Pre-culture of Gibson F1, F2 colonies</div> | |
- | + | ||
+ | <div id="content_note" > | ||
- | + | <b>Results</b></br></br> | |
+ | <span id="little">We checked that there were several colonies on the plate. 10 colonies for each fragment were picked, and culture into 3mL LB media containing 30ul Kanamycin.</br></br></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="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> | <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> | ||
</section> | </section> | ||
<section id="28"> | <section id="28"> | ||
<div class="date">September 28<sup>th</sup> 2012</div></br> | <div class="date">September 28<sup>th</sup> 2012</div></br> | ||
- | < | + | <img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></br> |
- | + | <div class="note-title">Vector preparation and PCR</div> | |
- | + | ||
+ | <div id="content_note" > | ||
- | + | <b>Results</b></br></br> | |
+ | <span id="little">For each cultured cells, vectors were mini-prepped, and pcr was done with proper primers (F1 : 1201 F, 1197R, F2 : 1198F, 1516R)</br></br></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="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> | <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> | ||
</section> | </section> |
Latest revision as of 03:59, 27 October 2012
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2012 KAIST Korea
Mail : kaist.igem.2012@gmail.com
Twitter : twitter.com/KAIST_iGEM_2012
Facebook : www.facebook.com/KAISTiGEM2012
Notebook : Labnote-September
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
![](https://static.igem.org/mediawiki/2012/9/95/Starter_gradient_kaist.png)
Labnote
SeptemberSeptember 1st 2012
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fdh pBAD, fdh pTrc colony PCR check
Results
Discussion
To check the presence of FDH gene, we picked the colony from the plate and did colony PCR. For all the colonies we picked, we confirmed that correct size of FDH is present.
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
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Bxb1_GTG_pBAD cloning with remaining DNAs
Remaining Bxb1_GTG insert was cloned into pBADmycHisC vector. And electrotransformed into MG1655.
Back to the CalendarSeptember 2nd 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Bxb1_ATG_pTrc, pBAD, Bxb1_GTG_pTrc miniprep for double transformation, Bxb1_GTG_pBAD colony inoculation and vector miniprep NcoI single cut check.
Results
Discussions
All the vectors showed right sizes. But Bxb1_GTG_pBAD colony #1 through 3 have undesired bands. They may be the supercoiled vector.
![2_0902Fig1](https://static.igem.org/mediawiki/2012/1/1c/KAIST_2_Sept.png)
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
Bxb1_ATG_pTrc, pBAD, Bxb1_GTG_pTrc double transformation into pPoC and pPoCpi
Back to the CalendarSeptember 3rd 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png)
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1201 1197 1198 1516 PCR for Gibson assembly.
Results
Discussion
There was no band for 1516 gene. Band of all three genes appeared at the right size.
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
1516 PCR for Gibson assembly.
Results
Discussion
Also for this time, there was no right size of band appeared for 1516 gene.
We,
1. changed the fraction of primers down to 0.5ul each, (lane 1)
2. changed the polymerase to pfu-x, (lane3)
3. used hybrid primers ( TOPO forward, Gibson reverse : lane 4 and TOPO reverse, Gibson forward : lane 5)
Lane 2 was the control experiment with Hotstartaq polymerase.
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
Pre-culture of piBR181 vector containing cells.
Procedure
We did 3ml pre-culture on five 10ml tubes for the cells containing each gene. Cell was grown about 16hours after pre-culture, and then induced with proper inducer.
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
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Bxb1 double transformants vector miniprep & PCR check
Results
Discussions
Seemed to every colonies have appropriate vectors.
Back to the CalendarSeptember 4th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png)
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1516 PCR for Gibson assembly.
Results
Discussion
Also for this time, there was no right size of band appeared for 1516 gene.
Back to the CalendarSeptember 5th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png)
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1201 1197 1198 PCR amplification
Results
Discussion
To amplify the amount of each fragment, we did PCR amplification of each gene once more. Correct size of bands appeared.
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
1197 1516 PCR amplification
Results
Discussion
To amplify the amount of each fragments, we did PCR amplification of 1197 once more. And we did PCR amplification of 1516 gene once more with the same primers. Correct size of bands appeared for 1197 gene, but there was no band for 1516 gene. .
Back to the CalendarSeptember 6th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png)
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1198 1516PCR amplification
Results
Discussion
To amplify the amount of each fragments, we did PCR amplification of 1198 once more. And we did PCR amplification of 1516 gene once more with the same primers. For 1516 gene, we couldn’t have right size of band for new primers. So we decided to use the fragments of 1516 that does not contain terminator sequence.
Back to the CalendarSeptember 7th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png)
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Pre-culture of 0109, 1191, FDH containing cells.
Discussion
We did 5ml pre-culture on 10ml tube for the cells containing each gene. Cell was grown about 16hours after pre-culture, and then induced with proper inducer.
Back to the Calendar
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
PACKMAN
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
PACKMAN
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
PACKMAN
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Flip Flop
Kaist Footer
September 8th 2012
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FDH pTrc & pBAD/0109 pBAD/1191 pBAD MG1655 expression check (sampling and induction)
Procedure
We seeded cells (500λ of cells put into 50mL LB with 50λ Ampicillin) and checked Optical Density. According to the O.D. value, we induced 37℃ and 30℃ sample and did sampling. Each samples were induced by different conditions ; 1mM and 2mM of IPTG, 10mM and 30mM of arabinose.
Back to the CalendarSeptember 9th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png)
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Moth_0109 expression optimization _ gel picture
Results
Discussion
For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 59.94kDa protein, formate--tetrahydrofolate ligase. The gel was ruined during staining/destaining procedure, and we couldn’t find any band for this protein.
Therefore, we concluded that expression of 0109 gene on pBAD vector has failed and decided to try once more.
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
Moth_1191 expression optimization _ gel picture
Results
Discussion
For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 33.92kDa protein, methylenetetrahydrofolate reductase. The gel was ruined during staining/destaining procedure, and we couldn’t find any band for this protein.
Therefore, we concluded that expression of 1191 gene on pBAD vector has failed and decided to try once more.
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
FDH pTrc expression optimization _ gel picture
Results
Discussion
For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 40.37kDa protein, Formate dehydrogenase. The gel was ruined during staining/destaining procedure, and we couldn’t find any band for this protein.
Therefore, we concluded that expression of FDH gene on pTrc vector has failed and decided to try once more.
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
FDH pBAD expression optimization _ gel picture
Results
Discussion
For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 40.37kDa protein, Formate dehydrogenase. The gel was ruined during staining/destaining procedure, and we couldn’t find any band for this protein.
Therefore, we concluded that expression of FDH gene on pTrc vector has failed and decided to try once more.
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
Primer design to PCR-amplify piBR181 vector.
Results
Discussion
For the Gibson assembly with RE digestion of vector, we had several problems with de-phosphorylation and purification after de-phosphorylation. Therefore we decided to PCR-amplify the piBR181 vector and use the PCR product in Gibson assembly.
Back to the CalendarSeptember 10th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png)
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PCR amplification of 1202 gene with new primers
Results
Discussion
The correct size of band appeared for the second PCR product, but there was no band appeared for the first PCR product.
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Bxb1_GTG_pBAD single cut check with different enzyme
Single cut with SnaBI
Results
No desired size(5.6kb) vector appeared.
Back to the Calendar
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
PACKMAN
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
PACKMAN
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
PACKMAN
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
PACKMAN
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
PACKMAN
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
PACKMAN
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
September 11th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
PCR amplification of piBR181 vector
Results
Discussion
The correct size of piBR181 vector pcr product is 5297bp. There appeared the right size and unspecific band on the gel. So we decided to do pcr once more.
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
Pre-culture of 0109, 1191, FDH containing cells.
Procedure
We did 5ml pre-culture on 10ml tube for the cells containing each gene. Cell was grown about 16hours after pre-culture, and then induced with proper inducer.
Back to the CalendarSeptember 12th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
PCR amplification of piBR181 vector : 10 sample
Results
Discussion
The full PCR construct of piBR181 vector is 5297bp. From the gel image, we could find that right size of piBR181 PCR product clearly exists.
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
FDH pTrc & pBAD/0109 pBAD/1191 pBAD MG1655 expression check (sampling and induction)
Procedure
We seeded cells (500λ of cells put into 50mL LB with 50λ Ampicillin) and checked Optical Density. According to the O.D. value, we induced 37℃ and 30℃ sample and did sampling. Each samples were induced by different conditions ; 1mM and 2mM of IPTG, 10mM and 30mM of arabinose.
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Bxb1_GTG_pBAD colony #4~7 inoculation, miniprep and single cut check
Single cut with SnaBI
Single cut with SnaBI
Results
Cloning completed
Back to the Calendar![2_0912Fig1](https://static.igem.org/mediawiki/2012/1/13/KAIST_12_Sept.png)
September 13th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Moth_0109 pBAD expression check
Results
Discussion
For each samples of different induction conditions, we ran 10% SDS-PAGE gel and checked the presence of 59.94kDa protein, formate--tetrahydrofolate ligase. We couldn’t find any band for this protein.
There was no significant band on the gel, so we couldn’t conclude that this protein is expressed. Our opinion is that we should run Western blot to exactly find this protein.
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
Moth_1191 pBAD expression check
Results
Discussion
For each samples of different induction conditions, we run 15% SDS-PAGE gel and checked the presence of 33.92kDa protein, methylenetetrahydrofolate reductase. Although we were not able to discriminate each band of insoluble fraction, We could find that there exists right band at the right size.
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
FDH pBAD expression check
Results
Discussion
For each samples of different induction conditions, we run 8% SDS-PAGE gel and checked the presence of 40.37 kDa protein, Formate dehydrogenase. We couldn’t find any band for this protein.
There was no significant band on the gel, so we couldn’t conclude that this protein is expressed. Our opinion is that we should run Western blot to exactly find this protein.
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
FDH pTrc expression check
Results
Discussion
For each samples of different induction conditions, we run 8% SDS-PAGE gel and checked the presence of 40.37 kDa protein, Formate dehydrogenase. We couldn’t find any band for this protein.
There was no significant band on the gel, so we couldn’t conclude that this protein is expressed. Our opinion is that we should run Western blot to exactly find this protein.
Back to the CalendarSeptember 14th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Gibson assembly – assembly of pcr fragments (pcr fragment of vector added)
Results
Among three methods to assemble DNA fragments, we chose third method, which is assembly of PCR amplified DNA fragments and vector.
0.17umoles of each fragments are added into the reaction sample, and volume to be added for each fragments has calculated on the assumption that average MW of each base is 660Da. Table below is the result of calculation.
After 1 hour of incubation on 50℃, the vector was transformed into TOP10 and NEB competent cell. Cells were plated and grown overnight during colony formation.
Back to the CalendarSeptember 15th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
BBa_C0060, BBa_C0061, BBa_C0062 transformation into TOP10
Templates for Auto-regulated FlipFlop project.
Back to the CalendarSeptember 16th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Gibson assembly colony pcr
Results
Discussion
As we couldn’t get full fragment by colony-pcr, we did 3ml culture, plasmid mini-prep and PCR for each colony. This will take one more day to get result.
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
BBa_C0060, BBa_C0061, BBa_C0062, bFMO inoculation and enzyme cut check
BBa_C0060, BBa_C0061, BBa_C0062: from colony, EcoRI single cut
Back to the Calendar- BBa_C0060: 2859bp
- BBa_C0061: 2688bp
- BBa_C0062: 2826bp
- bFMO: from cell stock, EcoRI single cut
September 17th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Gibson assembly fragment PCR after prep
Results
Discussion
There appeared correct size of band with several unspecific bands on the gel. We couldn’t even try to do gel extraction because the band was too thin and faint. We decided to do more rounds of pcr amplification.
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Template preparation for OE PCR
Promoter construct: 210bp
Results
Promoter PCR failed
Back to the Calendar
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
PACKMAN
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
PACKMAN
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
PACKMAN
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Flip Flop
September 18th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Template preparation for OE PCR
Results
Terminator PCR failed
Back to the CalendarSeptember 19th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Gibson F1 PCR after prep
Results
Discussion
We did PCR amplification after prep of F1 vector, but there appeared no band on the gel.
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
Gibson F2 5-1~5, F1-3-1~5, F1-4-1~5
Results
Discussion
There appeared several unspecific bands on the gel and
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Template preparation for OE PCR
Terminator: 179bp
Results
→ Gel extracted
Back to the CalendarSeptember 20th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Template preparation for OE PCR
Bxb1 Xis: 777bp
Results
bFMO PCR w/ various condtions
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
Bxb1_Xis-pGEM_B1 transfomation
Synthesized gene – Bxb1_Xis arrived and transformed into TOP10
Back to the CalendarSeptember 21st 2012
No Special Event!
Back to the CalendarSeptember 22nd 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Overlapping extension PCR
Results
→ Gel extracted
Back to the CalendarSeptember 23rd 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Overlapping extension PCR-Nested PCR
Results
Discussions
There was too little full construct nested. We think this is due to primer interfering by its LVA homology.
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
Overlapping extension PCR 2
Results
Discussions
Bands were more intense than those of first trial.
Back to the CalendarSeptember 24th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Overlapping extension PCR Gel Extraction
Results
Back to the Calendar![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
P1, PI construct
Results
Full fragment for pAuto is overlapped. The right size(3794bp) fragments are gel extracted.
September 25th 2012
No Special Event!
Back to the CalendarSeptember 26th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Full pAuto and pAutoSimple Overlapping PCR
Results
Discussion
The band intensity is too weak.
Back to the Calendar![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
pAutoIntegrase Overlapping PCR
Results
Full construct(2.5kb) for pAutoIntegrase is overlapped. The products are pcr purified and performed nested-pcr. 4th lane is the OE pcr product with rearranged template concentration.
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
Template preparation of terminator
Results
Terminator template for OE pcr is re-amplified with pfu-X DNA polymerase. The products are pcr purified.
September 27th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Pre-culture of Gibson F1, F2 colonies
Results
We checked that there were several colonies on the plate. 10 colonies for each fragment were picked, and culture into 3mL LB media containing 30ul Kanamycin.
Back to the CalendarSeptember 28th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Vector preparation and PCR
Results
For each cultured cells, vectors were mini-prepped, and pcr was done with proper primers (F1 : 1201 F, 1197R, F2 : 1198F, 1516R)
Back to the CalendarSeptember 29th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Template preparation of bFMO (for pAuto and pAutoSimple)
Results
To find out the right temperature for primer binding, we performed gradient pcr from 45℃~55℃.
But, all the pcr products showed non-specific binding of primers, and we selected the most proper temperature.
Back to the Calendar![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
Overlapping extension PCR Gel Extraction
Results
September 30th 2012
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
![](https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png)
![](https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png)
Template vector preparation – double digestion check with EcoRI and PstI
Results
Vector(BBa_J04450-pSB4A5) which will be used for cloning of pAutoIntegrase.
Back to the Calendar![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
bFMO template amplification with optimized condition
Results
![](https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png)
pAutoIntegrase cloning check
Results
PCR amplified from prepped vector