http://2014.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=20&target=Hwangas2014.igem.org - User contributions [en]2024-03-29T11:55:01ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Template:Team:Paris_Saclay/default_headerTemplate:Team:Paris Saclay/default header2015-08-07T06:37:44Z<p>Hwangas: Undo revision 406750 by Hwangas (talk)</p>
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<div id="pageContent"></html></div>Hwangashttp://2014.igem.org/Template:Team:Paris_Saclay/default_headerTemplate:Team:Paris Saclay/default header2015-08-07T06:34:56Z<p>Hwangas: </p>
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Every system that is genetically engineered harbors a potentially fatal vulnerability. The source of life's great diversity - spontaneous mutation - is for the synthetic biologist the source of constant apprehension and risk. The relentlessness of genetic mutation has discouraged attempts to treat it as anything other than unavoidable fact of tinkering with biology. Whether in a multifaceted genetic circuit or a simple protein expression platform, mutation is inevitable, and once it disrupts function, the organism will no longer experience the burden of transgene expression, causing the mutant to outcompete whatever has the intended sequence. Evolution and mutation work hand in hand to select against the maintenance of synthetic DNA sequences. Indeed, the mantra has been that time, in the form of mutation and evolution, will always find a way to erode and ultimately destroy everything that an engineer builds, no matter how ingeniously it may designed.<br />
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This year, here at Vanderbilt iGEM we are fighting back. We are proposing a novel approach based on rationally designed genomic architectures that promises to offer synthetic biologists unprecedented control over the evolutionary stability of their creations. At the heart of our strategy is an advanced computational algorithm that integrates decades worth of scientific data in order to identify and correct the highly-mutation prone 'hotspots' that lurk in every gene. Our strategy has a strong foundation in a rich literature from the fields of cancer biology and others that have annotated and characterized mutation hotspots for almost every conceivable source of mutagen, from ultraviolet radiation to recombination to polymerase errors. <br />
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When combined with synthetic DNA technology, our process becomes a simple and reliable optimization that is universally applicable to any coding gene being expressed in any organism. Our project first demonstrates the power of this rational synthetic gene design strategy by employing several canonical as well as highly original protocols for assaying DNA damage and its effects on the stability of artificial genetic elements. From these techniques, we can quantify everything from the selective subset of mutation types occurring on an in vitro level, up to how mutational loss of function translates at the scale of populations of genetically modified organisms.<br />
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To complement our work, we have harnessed our algorithm for use in what is becoming one of the most important tools for engineering biological molecules: directed evolution experiments. Not only can our engineered changes increase evolutionary stability in applications such as transgenic bioreactors, but it can also construct gene sequences that are more prone to mutate, thus accelerating studies into how to use evolutionary selection to produce tailored functional modifications to proteins.<br />
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Finally, we have investigated ways to build new genetically modified strains that exhibit greatly increased resistance to mutation. Combining our sequence-based strategies with the introduction of exogenous genes and removal of endogenous genes has enabled us to produce an expression platform for synthetic genes that not only has enhanced DNA repair mechanisms, but also has an entire artificial pathway introduced for the elimination of mutant strains from a population. <br />
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While any single engineered change to reduce mutation may still fail, when our innovative approaches to modulating evolutionary stability are taken in combination, they offer an unprecedented hope for taming evolutionary entropy. More than a victory for synthetic biology, we prove that through rational design principles- exactly what mutation most virulently tries to uproot- and with enough clever innovations, it is possible to defend against what seemed like an inevitability of nature. Score one for engineering.<br />
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</html></div>Hwangashttp://2014.igem.org/Team:Vanderbilt_MF/NotebookTeam:Vanderbilt MF/Notebook2015-02-08T21:21:53Z<p>Hwangas: </p>
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<div id="safety_tab_right" class = "tab_right"><a href="Safety"> </div></a><br />
<div id="attributions_tab_right" class = "tab_right"><a href="Attributions"> </div></a><br />
</div><br />
<br />
<div id="openLabBook"><br />
<div id="left_page" class="page"><br />
<header>Notebook</header><br />
<p>Notebook updates are displayed in lab-folder with exportation capabilities through Mendeley.</p><br />
</div><br />
<br />
<div id="right_page" class="page"><br />
</div><br />
</div><br />
<br />
<div id="left_button" class="button"></div><br />
<div id="right_button" class="button"></div><br />
</body><br />
<br />
</html></div>Hwangashttp://2014.igem.org/Team:Vanderbilt/SafetyTeam:Vanderbilt/Safety2015-02-08T21:19:27Z<p>Hwangas: </p>
<hr />
<div>{{CSS/Main}}<br />
<br />
<html><br />
<style type="text/css"><br />
body {<br />
position: relative;<br />
width: 850px //100%;<br />
margin: 0;<br />
padding: 0;<br />
padding-bottom: 10px;<br />
background:url(https://static.igem.org/mediawiki/parts/8/86/VU_Campus_photo_10.JPG);<br />
background-repeat: no-repeat;<br />
background-attachment: fixed;<br />
background-size:100% auto;<br />
font-family: Georgia, Times, "Times New Roman", serif; <br />
}<br />
<br />
table {<br />
cellpadding: 10;<br />
cellspacing:5;<br />
width: 75%;<br />
margin-left: auto; <br />
margin-right: auto;<br />
background-color: rgba(204,153,0,0.8);<br />
border-radius: 8px;<br />
height: 60px;"<br />
colspan: 2;<br />
padding: 5px;<br />
}<br />
<br />
p {<br />
font-size:1.25em;<br />
}<br />
<br />
.firstHeading { display: none;}<br />
.printfooter { display: none;}<br />
</style><br />
<br />
<center><img src="https://static.igem.org/mediawiki/parts/7/72/VU_iGem_Logo_%28Transparent.png" align="middle" width="500px" style="margin-left: 5px"></center><br />
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<!--main content --><br />
<table width="70%" align="center" margin-bottom: "3cm"; style="border:4px solid black;"<br />
style="margin: 1em auto 1em auto;"<br />
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<br />
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<td align="center" colspan="3"><br />
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<table width="100%" padding-bottom: "15"><br />
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<tr heigth="15px"></tr><br />
<tr heigth="75px"> <br />
<br />
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000> <br />
<a href="https://2014.igem.org/Team:Vanderbilt"style="color:#CC9900">Home<br><br />
<img src="https://static.igem.org/mediawiki/parts/c/cc/VU_vumc_home.jpg" width="150px"> </td> </a><br />
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<td style="border:1px solid black" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000> <br />
<a href="https://2014.igem.org/Team:Vanderbilt/Project"style="color:#CC9900">Project<br><br />
<img src="https://static.igem.org/mediawiki/parts/0/09/VU_Yeast_tubes_front.JPG" width="120px"> </td></a><br />
<br />
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000> <br />
<a href="https://2014.igem.org/Team:Vanderbilt/Parts"style="color:#CC9900">Parts</br><br />
<img src="https://static.igem.org/mediawiki/parts/8/8b/VU_transparent_pVU.gif" width="150px"> </td></a><br />
<br />
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000> <br />
<a href="https://2014.igem.org/Team:Vanderbilt/Notebook"style="color:#CC9900">Notebook<br><br />
<img src="https://static.igem.org/mediawiki/parts/c/ca/VU_sample_Lab_Journal_Page.JPG" width="90px"></td></a> <br />
<br />
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000> <br />
<a href="https://2014.igem.org/Team:Vanderbilt/Safety"style=" color:#CC9900">Safety</br><br />
<img src="https://static.igem.org/mediawiki/parts/e/eb/Safety_logo.gif" width="90px"></td></a><br />
<br />
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000> <br />
<a href="https://2014.igem.org/Team:Vanderbilt/Attributions"style="color:#CC9900">Attributions<br><br />
<img src="https://static.igem.org/mediawiki/parts/0/0e/Attributions_page_clipart.jpg" width="130px"></td></a><br />
<br />
<td align ="center"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="55px"></a> </td><br />
</tr><br />
</table><br />
<br />
</tr><br />
</tr><br />
</td><br />
<br />
<!--main content --><br />
<table width="70%" align="center"><br />
<tr><td><br />
<img src="https://static.igem.org/mediawiki/parts/7/7b/VU_lab_Layout_1.JPG" width="350px" align="right"><br />
<p><br />
Although our project did not use any particularly hazardous materials and both model organisms are classified in the lowest risk group, we took the safety of all our members as a high priority. Before being permitted to enter the lab, all members were required to pass a series of online safety training courses through Vanderbilt's VandySafe system. During a separate safety training meeting, members were informed of the proper way to dispose all used lab materials, procedures to take in the event of an emergency, proper handling of certain potentially hazardous chemicals, and general safe lab equipment use for items like Bunsen burners. We modeled our safety training after suggestions made by Vanderbilt Environmental Health and Safety (VEHS). <br />
</p><br />
<p><br />
One area of extra concern is that the transformed E. coli strains used have antibiotic resistance genes against both ampicillin and kanamycin. If these organisms were to escape the lab environment, it is conceivable that these resistance genes could undergo lateral gene transfer. The spread of antibiotic resistance in this way is significant to considerations of public health. Aside from the number of laboratory precautions we had taken in terms of maintaining sterile technique and properly disposing of all possible biohazards, in the design of our project we noted that homologous recombination into yeast would greatly reduce the danger of resistance genes spreading to other organisms.<br />
</p><br />
</d><br />
</tr><br />
</table></div>Hwangashttp://2014.igem.org/Team:Vanderbilt/SafetyTeam:Vanderbilt/Safety2015-02-08T21:19:06Z<p>Hwangas: </p>
<hr />
<div>{{CSS/Main}}<br />
<br />
<html><br />
<style type="text/css"><br />
body {<br />
position: relative;<br />
width: 850px //100%;<br />
margin: 0;<br />
padding: 0;<br />
padding-bottom: 10px;<br />
background:url(https://static.igem.org/mediawiki/parts/8/86/VU_Campus_photo_10.JPG);<br />
background-repeat: no-repeat;<br />
background-attachment: fixed;<br />
background-size:100% auto;<br />
font-family: Georgia, Times, "Times New Roman", serif; <br />
}<br />
<br />
table {<br />
cellpadding: 10;<br />
cellspacing:5;<br />
width: 75%;<br />
margin-left: auto; <br />
margin-right: auto;<br />
background-color: rgba(204,153,0,0.8);<br />
border-radius: 8px;<br />
height: 60px;"<br />
colspan: 2;<br />
padding: 5px;<br />
}<br />
<br />
p {<br />
font-size:1.25em;<br />
}<br />
<br />
.firstHeading { display: none;}<br />
.printfooter { display: none;}<br />
</style><br />
<br />
<center><img src="https://static.igem.org/mediawiki/parts/7/72/VU_iGem_Logo_%28Transparent.png" align="middle" width="500px" style="margin-left: 5px"></center><br />
<br />
<!--main content --><br />
<table width="70%" align="center" margin-bottom: "3cm"; style="border:4px solid black;"<br />
style="margin: 1em auto 1em auto;"<br />
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<tr> <br />
<br />
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<td align="center" colspan="3"><br />
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<table width="100%" padding-bottom: "15"><br />
<border-width=1px ><br />
<tr heigth="15px"></tr><br />
<tr heigth="75px"> <br />
<br />
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000> <br />
<a href="https://2014.igem.org/Team:Vanderbilt"style="color:#CC9900">Home<br><br />
<img src="https://static.igem.org/mediawiki/parts/c/cc/VU_vumc_home.jpg" width="150px"> </td> </a><br />
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<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000> <br />
<a href="https://2014.igem.org/Team:Vanderbilt/Team"style="color:#CC9900">Team</br><br />
<img src="https://static.igem.org/mediawiki/parts/d/d3/Lab_work_banner_image.JPG" width="120px"> </td> </a> <br />
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<td style="border:1px solid black" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000> <br />
<a href="https://2014.igem.org/Team:Vanderbilt/Project"style="color:#CC9900">Project<br><br />
<img src="https://static.igem.org/mediawiki/parts/0/09/VU_Yeast_tubes_front.JPG" width="120px"> </td></a><br />
<br />
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000> <br />
<a href="https://2014.igem.org/Team:Vanderbilt/Parts"style="color:#CC9900">Parts</br><br />
<img src="https://static.igem.org/mediawiki/parts/8/8b/VU_transparent_pVU.gif" width="150px"> </td></a><br />
<br />
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000> <br />
<a href="https://2014.igem.org/Team:Vanderbilt/Notebook"style="color:#CC9900">Notebook<br><br />
<img src="https://static.igem.org/mediawiki/parts/c/ca/VU_sample_Lab_Journal_Page.JPG" width="90px"></td></a> <br />
<br />
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000> <br />
<a href="https://2014.igem.org/Team:Vanderbilt/Safety"style=" color:#CC9900">Safety</br><br />
<img src="https://static.igem.org/mediawiki/parts/e/eb/Safety_logo.gif" width="90px"></td></a><br />
<br />
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000> <br />
<a href="https://2014.igem.org/Team:Vanderbilt/Attributions"style="color:#CC9900">Attributions<br><br />
<img src="https://static.igem.org/mediawiki/parts/0/0e/Attributions_page_clipart.jpg" width="130px"></td></a><br />
<br />
<td align ="center"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="55px"></a> </td><br />
</tr><br />
</table><br />
<br />
</tr><br />
</tr><br />
</td><br />
<br />
<!--main content --><br />
<table width="70%" align="center"><br />
<tr><td><br />
<img src="https://static.igem.org/mediawiki/parts/7/7b/VU_lab_Layout_1.JPG" width="350px" align="right"><br />
Although our project did not use any particularly hazardous materials and both model organisms are classified in the lowest risk group, we took the safety of all our members as a high priority. Before being permitted to enter the lab, all members were required to pass a series of online safety training courses through Vanderbilt's VandySafe system. During a separate safety training meeting, members were informed of the proper way to dispose all used lab materials, procedures to take in the event of an emergency, proper handling of certain potentially hazardous chemicals, and general safe lab equipment use for items like Bunsen burners. We modeled our safety training after suggestions made by Vanderbilt Environmental Health and Safety (VEHS). <br />
<br><br><br />
One area of extra concern is that the transformed E. coli strains used have antibiotic resistance genes against both ampicillin and kanamycin. If these organisms were to escape the lab environment, it is conceivable that these resistance genes could undergo lateral gene transfer. The spread of antibiotic resistance in this way is significant to considerations of public health. Aside from the number of laboratory precautions we had taken in terms of maintaining sterile technique and properly disposing of all possible biohazards, in the design of our project we noted that homologous recombination into yeast would greatly reduce the danger of resistance genes spreading to other organisms.<br />
</d><br />
</tr><br />
</table></div>Hwangashttp://2014.igem.org/Team:Vanderbilt/SafetyTeam:Vanderbilt/Safety2015-02-08T21:18:54Z<p>Hwangas: </p>
<hr />
<div>{{CSS/Main}}<br />
<br />
<html><br />
<style type="text/css"><br />
body {<br />
position: relative;<br />
width: 850px //100%;<br />
margin: 0;<br />
padding: 0;<br />
padding-bottom: 10px;<br />
background:url(https://static.igem.org/mediawiki/parts/8/86/VU_Campus_photo_10.JPG);<br />
background-repeat: no-repeat;<br />
background-attachment: fixed;<br />
background-size:100% auto;<br />
font-family: Georgia, Times, "Times New Roman", serif; <br />
}<br />
<br />
table {<br />
cellpadding: 10;<br />
cellspacing:5;<br />
width: 75%;<br />
margin-left: auto; <br />
margin-right: auto;<br />
background-color: rgba(204,153,0,0.8);<br />
border-radius: 8px;<br />
height: 60px;"<br />
colspan: 2;<br />
padding: 5px;<br />
}<br />
<br />
.firstHeading { display: none;}<br />
.printfooter { display: none;}<br />
</style><br />
<br />
<center><img src="https://static.igem.org/mediawiki/parts/7/72/VU_iGem_Logo_%28Transparent.png" align="middle" width="500px" style="margin-left: 5px"></center><br />
<br />
<!--main content --><br />
<table width="70%" align="center" margin-bottom: "3cm"; style="border:4px solid black;"<br />
style="margin: 1em auto 1em auto;"<br />
<br />
<tr> <br />
<br />
<!--navigation menu --><br />
<td align="center" colspan="3"><br />
<br />
<table width="100%" padding-bottom: "15"><br />
<border-width=1px ><br />
<tr heigth="15px"></tr><br />
<tr heigth="75px"> <br />
<br />
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000> <br />
<a href="https://2014.igem.org/Team:Vanderbilt"style="color:#CC9900">Home<br><br />
<img src="https://static.igem.org/mediawiki/parts/c/cc/VU_vumc_home.jpg" width="150px"> </td> </a><br />
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<a href="https://2014.igem.org/Team:Vanderbilt/Team"style="color:#CC9900">Team</br><br />
<img src="https://static.igem.org/mediawiki/parts/d/d3/Lab_work_banner_image.JPG" width="120px"> </td> </a> <br />
<br />
<td style="border:1px solid black" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000> <br />
<a href="https://2014.igem.org/Team:Vanderbilt/Project"style="color:#CC9900">Project<br><br />
<img src="https://static.igem.org/mediawiki/parts/0/09/VU_Yeast_tubes_front.JPG" width="120px"> </td></a><br />
<br />
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000> <br />
<a href="https://2014.igem.org/Team:Vanderbilt/Parts"style="color:#CC9900">Parts</br><br />
<img src="https://static.igem.org/mediawiki/parts/8/8b/VU_transparent_pVU.gif" width="150px"> </td></a><br />
<br />
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000> <br />
<a href="https://2014.igem.org/Team:Vanderbilt/Notebook"style="color:#CC9900">Notebook<br><br />
<img src="https://static.igem.org/mediawiki/parts/c/ca/VU_sample_Lab_Journal_Page.JPG" width="90px"></td></a> <br />
<br />
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000> <br />
<a href="https://2014.igem.org/Team:Vanderbilt/Safety"style=" color:#CC9900">Safety</br><br />
<img src="https://static.igem.org/mediawiki/parts/e/eb/Safety_logo.gif" width="90px"></td></a><br />
<br />
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000> <br />
<a href="https://2014.igem.org/Team:Vanderbilt/Attributions"style="color:#CC9900">Attributions<br><br />
<img src="https://static.igem.org/mediawiki/parts/0/0e/Attributions_page_clipart.jpg" width="130px"></td></a><br />
<br />
<td align ="center"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="55px"></a> </td><br />
</tr><br />
</table><br />
<br />
</tr><br />
</tr><br />
</td><br />
<br />
<!--main content --><br />
<table width="70%" align="center"><br />
<tr><td><br />
<img src="https://static.igem.org/mediawiki/parts/7/7b/VU_lab_Layout_1.JPG" width="350px" align="right"><br />
Although our project did not use any particularly hazardous materials and both model organisms are classified in the lowest risk group, we took the safety of all our members as a high priority. Before being permitted to enter the lab, all members were required to pass a series of online safety training courses through Vanderbilt's VandySafe system. During a separate safety training meeting, members were informed of the proper way to dispose all used lab materials, procedures to take in the event of an emergency, proper handling of certain potentially hazardous chemicals, and general safe lab equipment use for items like Bunsen burners. We modeled our safety training after suggestions made by Vanderbilt Environmental Health and Safety (VEHS). <br />
<br><br><br />
One area of extra concern is that the transformed E. coli strains used have antibiotic resistance genes against both ampicillin and kanamycin. If these organisms were to escape the lab environment, it is conceivable that these resistance genes could undergo lateral gene transfer. The spread of antibiotic resistance in this way is significant to considerations of public health. Aside from the number of laboratory precautions we had taken in terms of maintaining sterile technique and properly disposing of all possible biohazards, in the design of our project we noted that homologous recombination into yeast would greatly reduce the danger of resistance genes spreading to other organisms.<br />
</d><br />
</tr><br />
</table></div>Hwangashttp://2014.igem.org/Team:Vanderbilt/NotebookTeam:Vanderbilt/Notebook2015-02-08T21:18:28Z<p>Hwangas: </p>
<hr />
<div>{{CSS/Main}}<br />
<br />
<html><br />
<style type="text/css"><br />
body {<br />
position: relative;<br />
width: 850px //100%;<br />
margin: 0;<br />
padding: 0;<br />
padding-bottom: 10px;<br />
background:url(https://static.igem.org/mediawiki/parts/8/86/VU_Campus_photo_10.JPG);<br />
background-repeat: no-repeat;<br />
background-attachment: fixed;<br />
background-size:100% auto;<br />
font-family: Georgia, Times, "Times New Roman", serif; <br />
}<br />
<br />
table {<br />
cellpadding: 10;<br />
cellspacing: 5;<br />
width: 75%;<br />
margin-left: auto; <br />
margin-right: auto;<br />
background-color: rgba(204,153,0,0.8);<br />
border-radius: 8px;<br />
height: 60px;<br />
padding:5px;<br />
}<br />
<br />
td {<br />
colspan: 2<br />
}<br />
<br />
li {<br />
font-size:1.25em;<br />
}<br />
<br />
.firstHeading { display: none;}<br />
.printfooter { display: none; }<br />
<br />
</style><br />
<br />
<center><img src="https://static.igem.org/mediawiki/parts/7/72/VU_iGem_Logo_%28Transparent.png" align="middle" width="500px"></center><br />
<!--main content --><br />
<table width="70%" align="center" margin-bottom: "3cm"; style="border:4px solid black;"<br />
style="margin: 1em auto 1em auto;"<br />
<br />
<tr> <br />
<br />
<!--navigation menu --><br />
<td align="center" colspan="3"><br />
<br />
<table width="100%" padding-bottom: "15"><br />
<border-width=1px ><br />
<tr heigth="15px"></tr><br />
<tr heigth="75px"> <br />
<br />
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000> <br />
<a href="https://2014.igem.org/Team:Vanderbilt"style="color:#CC9900">Home<br><br />
<img src="https://static.igem.org/mediawiki/parts/c/cc/VU_vumc_home.jpg" width="150px"> </td> </a><br />
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<a href="https://2014.igem.org/Team:Vanderbilt/Team"style="color:#CC9900">Team</br><br />
<img src="https://static.igem.org/mediawiki/parts/d/d3/Lab_work_banner_image.JPG" width="120px"> </td> </a> <br />
<br />
<td style="border:1px solid black" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000> <br />
<a href="https://2014.igem.org/Team:Vanderbilt/Project"style="color:#CC9900">Project<br><br />
<img src="https://static.igem.org/mediawiki/parts/0/09/VU_Yeast_tubes_front.JPG" width="120px"> </td></a><br />
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<table width="70%" align="center"><br />
<tr><td colspan="3"> <h3 align="left"><font size="4">Lab Notebook</font></h3></td></tr><br />
<br />
<tr><br />
<td width="45%" valign="top"><br />
<br><br />
<br />
<p><b><font size="4">Spring 2014</font></b></p><br />
<img src="https://static.igem.org/mediawiki/parts/0/00/VU_Genomic_DNA_bands_check_LNS.JPG" align="right" width="250" alt="An example of a gel showing high molecular weight (>10 kb) bands corresponding to successfully extracted plant Genomic DNA (in this case, samples LNS1 and LNS2 from Arabidopsis DNA)" style="padding-bottom:0.5em;"><br />
<br />
<p><b>March 27<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Extracted genomic DNA from 100 mg of <i>Arabidopsis thaliana </i> leaves using a Viogene DNA extraction kit. Samples were labeled ZIN for zingiberene.<br />
</li><br />
<li><br />
Two samples were prepared and nanodropped. The concentration of the first was 2.6 ng/ul, and the second was 3.5 ng/ul of DNA, indicating a minimal yield of DNA. <br />
</li><br />
</ul><br />
<br />
<p><b>March 30<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Extracted genomic DNA from 100 mg of <i> Picea abies </i> needles using the same extraction kit and protocol. Samples were labeled CAR for carene.<br />
</li><br />
<li><br />
Two samples were prepared and nanodropped. The concentration of the first was 0.9 ng/ul, and the second was 3.5 ng/ul of DNA, indicating a minimal yield of DNA. <br />
</li><br />
</ul><br />
<p><b>March 31<sup>st</sup></b></p><br />
<ul><br />
<li><br />
Ran a 0.6 % argarose gel on the DNA extracted from ZIN and CAR, as well as the column flow-through from the kit. <br />
</li><br />
<li><br />
ZIN1, ZIN2, CAR1, and CAR2 all show a faint but distinct DNA band above the highest rung on the DNA ladder (>10kb), showing the presence of DNA. No bands were seen on the kit flow-through. Indicates successful genomic extraction. <br />
</li><br />
<li><br />
Preformed a second genomic extraction on <i>Picea abies </i> to improve yield. Nanodrop shows CAR3 to be at 6.2 ng/ul and CAR4 to be 11.5 ng/ul.<br />
</li><br />
<li><br />
Extracted genomic DNA from <i> Gossypium hirsutum </i>. Samples nanodroppped: CAD1 1.8 ng/ul and CAD2 7.8 ng/ul. <br />
</li><br />
</ul><br />
<br />
<p><b>April 1<sup>st</sup></b></p><br />
<ul><br />
<li><br />
Ran a gel on CAR3, CAR4, CAD1, and CAD2. Brighter genomic DNA bands were seen on the cadinene camples than before, but cadinene samples showed significant smearing near the top of the gel.<br />
</li><br />
</ul><br />
<br />
<p><b>April 2<sup>nd</sup></b></p><br />
<ul><br />
<li><br />
Extracted genomic DNA from <i>Salvia officinalis</i>. Samples nanodropped: SAB1 7.5 ng/ul, and SAB 11.4 ng/ul.<br />
</li><br />
<li><br />
Extracted genomic DNA from <i>Mentha citrata</i>. Samples nanodropped: LNR1 3.4 ng/ul, LNR2 6.2 ng/ul, LNR3 7.3 ng/ul<br />
</li><br />
<li><br />
E. coli containing p404GALS and pDZ207 from Addgene were grown on LB plates with ampicilin. These were miniprepped using a Viogen kit. Samples nanodropped: p404GALS (A) 130.2 ng/ul, p404GALS (B) 112.3 ng/ul, pDZ207 (A) 145.3 ng/ul, pDZ207 (B) 84.1 ng/ul. <br />
</li><br />
</ul><br />
<br />
<p><b>April 3<sup>rd</sup></b></p><br />
<ul><br />
<li><br />
Extracted genomic DNA again from <i> Arabidopsis thaliana </i>. Sample nanodropped: LNS1 53.4 ng/ul, LNS2 585.2 ng/ul<br />
</li><br />
<li><br />
Ran gel on LNR1, LNR2, LNR3, LNS1, LNS2, SAB1, and SAB2. All show high weight DNA bands, with Linalool (S) samples having the brightest.<br />
</li><br />
</ul><br />
<br />
<div style="width:300; font-size:80%; text-align:center;"><br />
<img src="https://static.igem.org/mediawiki/parts/5/5f/VU_humelene_genomic_dna_pcr.JPG" align=right width="250" alt="An example of a gel showing high molecular weight (>10 kb) bands corresponding to successfully extracted plant Genomic DNA (in this case, samples LNS1 and LNS2 from Arabidopsis DNA)" style="float:right; padding-bottom:0.5em;"></div><br />
<br />
<p><b>April 4<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Extracted genomic DNA from <i> Zingiber zermbet </i>. Sample nanodropped: HUM1 31.9 ng/ul and HUM2 18.6 ng/ul<br />
</li><br />
<li><br />
Extracted genomic DNA from <i> Ocimum basilicum </i>. Sample nanodropped: GER1 2.3 ng/ul<br />
</li><br />
</ul><br />
<br />
<p><b>April 5<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Extracted genomic DNA from <i> Santalum album </i> seeds since the sapling was still not fully grown. Sample nanodropped: SAN1 53.8 ng/ul and SAN2 28.4 ng/ul<br />
</li><br />
</ul><br />
<br />
<p><b>April 7<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Extracted genomic DNA from <i> Perilla frutescens </i>. Sample nanodropped: MYR1 632.8 ng/ul and MYR2 958.6 ng/ul<br />
</li><br />
<li><br />
Ran gel on MYR1, MYR2, SAN1, and SAN2. Myrcene samples show bright band at high weight as well as smearing toward bottom. Santelene samples have visible but much fainter genomic DNA band. <br />
</li><br />
</ul><br />
<br />
<p><b>April 24<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Preformed PCR using zingiberene synthase primers on LNS2 genomic DNA and using linalool (S) synthase primers on the sample template. <br />
</li><br />
<li><br />
Ran gel on PCR product. Resulted in no visible bands formed. <br />
</li><br />
</ul><br />
<br />
<p><b>April 25<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Took 1.8 ul of HUM1 genomic DNA and preformed a PCR with humelene synthase primers. <br />
</li><br />
<li><br />
Ran gel on PCR product. Resulted in three total bands, one faint around 1.2 kb, and two bands very close in size just under 3.0 kb. <br />
</li><br />
<li><br />
Both ~3 kb bands were gel extracted, combining across all four lanes. Samples nanodropped: HUM-top 8.5 ng/ul, HUM-bottom 10.6 ng/ul. <br />
</li><br />
</ul><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/e/e8/VU_labeled_PCON_Gel.jpg" align="right" width="350" ><br />
<br />
<p><b>April 27<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Preformed PCR using linalool (R) synthase primers on LNR3 genomic DNA. <br />
</li><br />
<li><br />
Ran gel on PCR product. Resulted in no visible bands formed. <br />
</li><br />
</ul><br />
<br />
<p><b>April 29<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Preformed PCR using myrcene synthase primers on MYR2 genomic DNA. <br />
</li><br />
<li><br />
Ran gel on PCR product. Smeared bands on gel but no distinct bands. <br />
</li><br />
<li><br />
Preformed overlap-extension PCR on the gel extracted humelene samples (both HUM-top and HUM-bottom) to add epitope tag sequence<br />
</li><br />
<li><br />
Ran gel on OE-PCR product. Only extremely faint bands were visible. <br />
</li><br />
</ul><br />
<br />
<br><br />
<br />
<b><font size="4">Summer 2014</font></b><br />
<br />
<p><b>May</b></p><br />
<ul><br />
<li><br />
Continued troubleshooting PCR reaction conditions for all of the terpenes that failed to amplify. Tried adjusting template concentration, adding DMSO, changing thermocycler program, hot start PCR, new polymerase and dNTPs<br />
</li><br />
<li><br />
Re-did genomic extractions for those that produced less than 50 ng/ul of genomic DNA. Used this new template in further PCRs<br />
</li><br />
<li><br />
Extracted the Gal10 gene from our template plasmid and a kanomycin resistance gene bordered by LoxP sites.<br />
</li><br />
</ul><br />
<br />
<p><b>June</b></p><br />
<ul><br />
<li><br />
All of the gene cassettes for plasmid construction that were successfully extracted so far were ligated together and inserted into the MCS of pUC19. This formed our first intermediate plasmid.<br />
</li><br />
</ul><br />
<br />
<br />
<br />
<p><b>July</b></p><br />
<ul><br />
<li><br />
Finally reached the point that all terpene genes were consistently amplifying with the synthase gene primers. The genomic DNA sample for sabinene was completely used before this point, although all other of the 8 terpenes showed clear bands.<br />
</li><br />
<br><br />
<img src="https://static.igem.org/mediawiki/parts/d/d5/VU_genomic_DNA_PCR.jpg" align="right" width="300" ><br />
<li><br />
The results of the genomic DNA PCR indicated each gene had a large fraction of introns. None of the genes had a distinct band at exactly the right weight corresponding with what the intron-less cDNA size would be.<br />
</li><br />
</ul><br />
<br />
<br><br />
<b><font size="4">Fall 2014</font></b><br />
<p><b>August</b></p><br />
<ul><br />
<li><br />
Moved the lab into its new space before the start of the semester<br />
</li><br />
<br />
<li><br />
Created the plasmid intermediate pVU1400A, which is missing only a single insert to become pVU14004.<br />
</li><br />
</ul><br />
<br />
<p><b>September</b></p><br />
<ul><br />
<li><br />
Ran RNA extraction on all the plants that were still available. This excluded Myrcene and Linalool (R) since both <i>Perilla frutescens</i> and <i> Mentha aquatica</i> had withered over the summered. <br />
</li><br />
<li><br />
Repeated RNA extractions on those which showed appreciable concentration on the nanodrop. Eventually all 7 remanining terpenes had plant RNA in appreciable quantities (most between 20-50 ng/ul, with a few less than 10 and a few more than 100 ng/ul).<br />
</li><br />
</ul><br />
<br />
<p><b>September 17<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Digested plasmid intermediate grown in demethylated bacteria with ClaI. Ligated final insert into vector. No transfomants grow after 24 hours. <br />
</li><br />
<li><br />
Diagnostic digest shows the ClaI enzyme is cutting properly. pUC19 positive control for transformations show that the competent cells are working. <br />
</li><br />
</ul><br />
<br />
<p><b>September 18<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Ran reverse transcription PCR on extracted RNA to isolate synthase cDNA. Humelene and sabinene show clear positive results, santalene shows amplification at smaller region, and cadinene shows no cDNA bands. <br />
</li><br />
</ul><br />
<br />
<p><b>September 19<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Made liquid cultures of K546546 in preparation for mutagenesis. Also made glycerol stock to store at -80. <br />
</li><br />
<li><br />
Diagnostic digest of ligation of pVU1400A intermediate and the final insert needed to make finished plasmid. Gel clearly shows bands in exactly the correct positions for each of three comparison conditions, proving that the creation of pVU14004 was finally successful. <br />
</li><br />
</ul><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/a/a7/VU_PVU14004_Conf_9_19.jpg" align="right" width="250"><br />
<br />
<p><b>September 20<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Miniprep of K546546 liquid cultures (1 ml ). First culture tube concentration of 85.7 ng/ul DNA, second 105.1 ng/ul<br />
</li><br />
<li><br />
RNA extracted arabadopsis and <i> Picea abies </i> to improve yield and quality. Carene still failed to get an RNA concentration greater than 10 ng/ul, while Arabidopsis produced 107 ng/ul with a good A260/A280 ratio. <br />
</li><br />
</ul><br />
<br />
<p><b>September 20<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Miniprep of K546546 liquid cultures (1 ml). First culture tube concentration of 85.7 ng/ul DNA, second 105.1 ng/ul<br />
</li><br />
</ul><br />
<br />
<p><b>September 26<sup>th</sup></b></p><br />
<ul><br />
<li><br />
RT-PCR done on humelene, linalool (S), sabinene, and zingiberene. Sabinene produces clear bands, while zingiberene shows one faint band at roughly the correct size. Positive controls are also run to confirm that the reverse transcription step is not the cause of any failures to amplify. <br />
</li><br />
</ul><br />
<br />
<p><b>October 5<sup>th</sup></b></p><br />
<ul><br />
<li><br />
RT-PCR done on humelene, sabinene, and santalene. Sabinene again produces clear bands,and santalene does as well although much <br />
fainter. Both bands were gel extracted to yield a small (<10 ng/ul) amount of DNA.<br />
</li><br />
<li><br />
Extracted DNA was ligated into pUC19 and transformed into E. coli.<br />
</li><br />
</ul><br />
<br />
<p><b>October 7<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Site direction mutagenesis kit and specially designed primers were used to mutagenize K546546 at its BglI site, sabinene cDNA at <br />
its XbaI and EcoRI sites, and pVU14004 at its EcoRI and XbaI sites. <br />
</li><br />
<li><br />
Mutagenesis product was transformed into E. coli.<br />
</li><br />
</ul><br />
<br />
<p><b>October 9<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Minipreps were done on 5 ml liquid cultures of mutagenized pVU14004, sabinene, and K546546. 4 liquid cultures were made for each, and both sabinene and pVU14004 were done in duplicate. <br />
</li><br />
<li><br />
Diagnostic digests were done on miniprepped plasmid to check if the restriction sites were mutagenized. pVU14004 appeared to have lost its XbaI site but not its EcoRI site, sabinene shows a size that suggests it failed to ligate as an insert into pUC19, and K546 shows only a single band at around its starting weight.<br />
</li><br />
</ul><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/d/d9/VU_Diagnostic_Digest_10-9.JPG" align="right" width="320"><br />
<br />
<p><b>October 10<sup>th</sup></b></p><br />
<ul><br />
<li><br />
All 8 minipreps of sabinene ligated into pUC19 were digested with SpeI and ApaI to check for the synthase insert. Only one, Sab B2, shows a second band at the right size.<br />
</li><br />
</ul><br />
<br />
<p><b>October 11<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Santalene synthase was ligated into pVU14004 and pSB1C3. E. coli was transformed and incubated.<br />
</li><br />
<li><br />
The sites that failed to show mutagenesis were mutagenized again using and transformed into E. coli. K546546 had its AgeI site mutagenized. <br />
</li><br />
</ul><br />
<br />
<p><b>October 12<sup>th</sup></b></p><br />
<ul><br />
<li><br />
All liquid cultures were miniprepped, producing 8 samples of pVU14004 with confirmed XbaI mutagenesis, 4 pVU14004 with no sites confirmed, 4 sabinene, and 6 samples produced from santalene in pVU14004. None of the plates with santalene in pSB1C3 produced colonies.<br />
</li><br />
<li><br />
Diagnostic digests were run on all miniprepped plasmid (K546- AgeI, BglI, SphI. Sabinene- EcoRI, BamH1. Santalene in pVU- ApaI, XbaI. pVU- EcoRI, XbaI, BamHI, KpnI). Santalene appeared not to have ligated into pVU14004. Sabinene did not have its EcoRI site removed by mutagenesis. K546546 had at least one cut, but the second sample may have had one site mutagenized. <br />
</li><br />
<li><br />
Santalene was re-ligated into pSB1C3 and transformed. <br />
</li><br />
</ul><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/5/53/VU_10-11_diagnostic_digest.JPG" align="right" width="250" ><br />
<br />
<p><b>October 13<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Santalene in pSB1C3 was miniprepped to good yield. Each of 4 replicates was digested with SpeI and ApaI to test for ligation. The ApaI enzyme appears not have cut, but the fourth sample showed an uncut plasmid size which corresponded to that of pSB1C with santalene successfully inserted. <br />
</li><br />
</ul><br />
<br />
<br />
<p><b>October 14<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Ligated santalene synthase again into pVU14004 and transformed into E. coli<br />
</li><br />
</ul><br />
<br />
<p><b>October 15<sup>th</sup></b></p><br />
<ul><br />
<li><br />
One colony grew and was put in liquid culture. <br />
</li><br />
<li><br />
Culture miniprepped and digested. Again no gene insertion was detectable.<br />
</li><br />
</ul><br />
<br />
<p><b>October 16<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Transformed pVU14004 into a dam- strain of E. coli to address the methylation sensitivity of ApaI. <br />
</li><br />
<li><br />
Finished planning and acquiring materials for GC-MS confirmation of terpene presence.<br />
</li><br />
</ul><br />
<br><br />
<br />
</html></div>Hwangashttp://2014.igem.org/Team:Vanderbilt/NotebookTeam:Vanderbilt/Notebook2015-02-08T21:18:12Z<p>Hwangas: </p>
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<tr><td colspan="3"> <h3 align="left"><font size="4">Lab Notebook</font></h3></td></tr><br />
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<tr><br />
<td width="45%" valign="top"><br />
<br><br />
<br />
<p><b><font size="4">Spring 2014</font></b></p><br />
<img src="https://static.igem.org/mediawiki/parts/0/00/VU_Genomic_DNA_bands_check_LNS.JPG" align="right" width="250" alt="An example of a gel showing high molecular weight (>10 kb) bands corresponding to successfully extracted plant Genomic DNA (in this case, samples LNS1 and LNS2 from Arabidopsis DNA)" style="padding-bottom:0.5em;"><br />
<br />
<p><b>March 27<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Extracted genomic DNA from 100 mg of <i>Arabidopsis thaliana </i> leaves using a Viogene DNA extraction kit. Samples were labeled ZIN for zingiberene.<br />
</li><br />
<li><br />
Two samples were prepared and nanodropped. The concentration of the first was 2.6 ng/ul, and the second was 3.5 ng/ul of DNA, indicating a minimal yield of DNA. <br />
</li><br />
</ul><br />
<br />
<p><b>March 30<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Extracted genomic DNA from 100 mg of <i> Picea abies </i> needles using the same extraction kit and protocol. Samples were labeled CAR for carene.<br />
</li><br />
<li><br />
Two samples were prepared and nanodropped. The concentration of the first was 0.9 ng/ul, and the second was 3.5 ng/ul of DNA, indicating a minimal yield of DNA. <br />
</li><br />
</ul><br />
<p><b>March 31<sup>st</sup></b></p><br />
<ul><br />
<li><br />
Ran a 0.6 % argarose gel on the DNA extracted from ZIN and CAR, as well as the column flow-through from the kit. <br />
</li><br />
<li><br />
ZIN1, ZIN2, CAR1, and CAR2 all show a faint but distinct DNA band above the highest rung on the DNA ladder (>10kb), showing the presence of DNA. No bands were seen on the kit flow-through. Indicates successful genomic extraction. <br />
</li><br />
<li><br />
Preformed a second genomic extraction on <i>Picea abies </i> to improve yield. Nanodrop shows CAR3 to be at 6.2 ng/ul and CAR4 to be 11.5 ng/ul.<br />
</li><br />
<li><br />
Extracted genomic DNA from <i> Gossypium hirsutum </i>. Samples nanodroppped: CAD1 1.8 ng/ul and CAD2 7.8 ng/ul. <br />
</li><br />
</ul><br />
<br />
<p><b>April 1<sup>st</sup></b></p><br />
<ul><br />
<li><br />
Ran a gel on CAR3, CAR4, CAD1, and CAD2. Brighter genomic DNA bands were seen on the cadinene camples than before, but cadinene samples showed significant smearing near the top of the gel.<br />
</li><br />
</ul><br />
<br />
<p><b>April 2<sup>nd</sup></b></p><br />
<ul><br />
<li><br />
Extracted genomic DNA from <i>Salvia officinalis</i>. Samples nanodropped: SAB1 7.5 ng/ul, and SAB 11.4 ng/ul.<br />
</li><br />
<li><br />
Extracted genomic DNA from <i>Mentha citrata</i>. Samples nanodropped: LNR1 3.4 ng/ul, LNR2 6.2 ng/ul, LNR3 7.3 ng/ul<br />
</li><br />
<li><br />
E. coli containing p404GALS and pDZ207 from Addgene were grown on LB plates with ampicilin. These were miniprepped using a Viogen kit. Samples nanodropped: p404GALS (A) 130.2 ng/ul, p404GALS (B) 112.3 ng/ul, pDZ207 (A) 145.3 ng/ul, pDZ207 (B) 84.1 ng/ul. <br />
</li><br />
</ul><br />
<br />
<p><b>April 3<sup>rd</sup></b></p><br />
<ul><br />
<li><br />
Extracted genomic DNA again from <i> Arabidopsis thaliana </i>. Sample nanodropped: LNS1 53.4 ng/ul, LNS2 585.2 ng/ul<br />
</li><br />
<li><br />
Ran gel on LNR1, LNR2, LNR3, LNS1, LNS2, SAB1, and SAB2. All show high weight DNA bands, with Linalool (S) samples having the brightest.<br />
</li><br />
</ul><br />
<br />
<div style="width:300; font-size:80%; text-align:center;"><br />
<img src="https://static.igem.org/mediawiki/parts/5/5f/VU_humelene_genomic_dna_pcr.JPG" align=right width="250" alt="An example of a gel showing high molecular weight (>10 kb) bands corresponding to successfully extracted plant Genomic DNA (in this case, samples LNS1 and LNS2 from Arabidopsis DNA)" style="float:right; padding-bottom:0.5em;"></div><br />
<br />
<p><b>April 4<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Extracted genomic DNA from <i> Zingiber zermbet </i>. Sample nanodropped: HUM1 31.9 ng/ul and HUM2 18.6 ng/ul<br />
</li><br />
<li><br />
Extracted genomic DNA from <i> Ocimum basilicum </i>. Sample nanodropped: GER1 2.3 ng/ul<br />
</li><br />
</ul><br />
<br />
<p><b>April 5<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Extracted genomic DNA from <i> Santalum album </i> seeds since the sapling was still not fully grown. Sample nanodropped: SAN1 53.8 ng/ul and SAN2 28.4 ng/ul<br />
</li><br />
</ul><br />
<br />
<p><b>April 7<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Extracted genomic DNA from <i> Perilla frutescens </i>. Sample nanodropped: MYR1 632.8 ng/ul and MYR2 958.6 ng/ul<br />
</li><br />
<li><br />
Ran gel on MYR1, MYR2, SAN1, and SAN2. Myrcene samples show bright band at high weight as well as smearing toward bottom. Santelene samples have visible but much fainter genomic DNA band. <br />
</li><br />
</ul><br />
<br />
<p><b>April 24<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Preformed PCR using zingiberene synthase primers on LNS2 genomic DNA and using linalool (S) synthase primers on the sample template. <br />
</li><br />
<li><br />
Ran gel on PCR product. Resulted in no visible bands formed. <br />
</li><br />
</ul><br />
<br />
<p><b>April 25<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Took 1.8 ul of HUM1 genomic DNA and preformed a PCR with humelene synthase primers. <br />
</li><br />
<li><br />
Ran gel on PCR product. Resulted in three total bands, one faint around 1.2 kb, and two bands very close in size just under 3.0 kb. <br />
</li><br />
<li><br />
Both ~3 kb bands were gel extracted, combining across all four lanes. Samples nanodropped: HUM-top 8.5 ng/ul, HUM-bottom 10.6 ng/ul. <br />
</li><br />
</ul><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/e/e8/VU_labeled_PCON_Gel.jpg" align="right" width="350" ><br />
<br />
<p><b>April 27<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Preformed PCR using linalool (R) synthase primers on LNR3 genomic DNA. <br />
</li><br />
<li><br />
Ran gel on PCR product. Resulted in no visible bands formed. <br />
</li><br />
</ul><br />
<br />
<p><b>April 29<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Preformed PCR using myrcene synthase primers on MYR2 genomic DNA. <br />
</li><br />
<li><br />
Ran gel on PCR product. Smeared bands on gel but no distinct bands. <br />
</li><br />
<li><br />
Preformed overlap-extension PCR on the gel extracted humelene samples (both HUM-top and HUM-bottom) to add epitope tag sequence<br />
</li><br />
<li><br />
Ran gel on OE-PCR product. Only extremely faint bands were visible. <br />
</li><br />
</ul><br />
<br />
<br><br />
<br />
<b><font size="4">Summer 2014</font></b><br />
<br />
<p><b>May</b></p><br />
<ul><br />
<li><br />
Continued troubleshooting PCR reaction conditions for all of the terpenes that failed to amplify. Tried adjusting template concentration, adding DMSO, changing thermocycler program, hot start PCR, new polymerase and dNTPs<br />
</li><br />
<li><br />
Re-did genomic extractions for those that produced less than 50 ng/ul of genomic DNA. Used this new template in further PCRs<br />
</li><br />
<li><br />
Extracted the Gal10 gene from our template plasmid and a kanomycin resistance gene bordered by LoxP sites.<br />
</li><br />
</ul><br />
<br />
<p><b>June</b></p><br />
<ul><br />
<li><br />
All of the gene cassettes for plasmid construction that were successfully extracted so far were ligated together and inserted into the MCS of pUC19. This formed our first intermediate plasmid.<br />
</li><br />
</ul><br />
<br />
<br />
<br />
<p><b>July</b></p><br />
<ul><br />
<li><br />
Finally reached the point that all terpene genes were consistently amplifying with the synthase gene primers. The genomic DNA sample for sabinene was completely used before this point, although all other of the 8 terpenes showed clear bands.<br />
</li><br />
<br><br />
<img src="https://static.igem.org/mediawiki/parts/d/d5/VU_genomic_DNA_PCR.jpg" align="right" width="300" ><br />
<li><br />
The results of the genomic DNA PCR indicated each gene had a large fraction of introns. None of the genes had a distinct band at exactly the right weight corresponding with what the intron-less cDNA size would be.<br />
</li><br />
</ul><br />
<br />
<br><br />
<b><font size="4">Fall 2014</font></b><br />
<p><b>August</b></p><br />
<ul><br />
<li><br />
Moved the lab into its new space before the start of the semester<br />
</li><br />
<br />
<li><br />
Created the plasmid intermediate pVU1400A, which is missing only a single insert to become pVU14004.<br />
</li><br />
</ul><br />
<br />
<p><b>September</b></p><br />
<ul><br />
<li><br />
Ran RNA extraction on all the plants that were still available. This excluded Myrcene and Linalool (R) since both <i>Perilla frutescens</i> and <i> Mentha aquatica</i> had withered over the summered. <br />
</li><br />
<li><br />
Repeated RNA extractions on those which showed appreciable concentration on the nanodrop. Eventually all 7 remanining terpenes had plant RNA in appreciable quantities (most between 20-50 ng/ul, with a few less than 10 and a few more than 100 ng/ul).<br />
</li><br />
</ul><br />
<br />
<p><b>September 17<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Digested plasmid intermediate grown in demethylated bacteria with ClaI. Ligated final insert into vector. No transfomants grow after 24 hours. <br />
</li><br />
<li><br />
Diagnostic digest shows the ClaI enzyme is cutting properly. pUC19 positive control for transformations show that the competent cells are working. <br />
</li><br />
</ul><br />
<br />
<p><b>September 18<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Ran reverse transcription PCR on extracted RNA to isolate synthase cDNA. Humelene and sabinene show clear positive results, santalene shows amplification at smaller region, and cadinene shows no cDNA bands. <br />
</li><br />
</ul><br />
<br />
<p><b>September 19<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Made liquid cultures of K546546 in preparation for mutagenesis. Also made glycerol stock to store at -80. <br />
</li><br />
<li><br />
Diagnostic digest of ligation of pVU1400A intermediate and the final insert needed to make finished plasmid. Gel clearly shows bands in exactly the correct positions for each of three comparison conditions, proving that the creation of pVU14004 was finally successful. <br />
</li><br />
</ul><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/a/a7/VU_PVU14004_Conf_9_19.jpg" align="right" width="250"><br />
<br />
<p><b>September 20<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Miniprep of K546546 liquid cultures (1 ml ). First culture tube concentration of 85.7 ng/ul DNA, second 105.1 ng/ul<br />
</li><br />
<li><br />
RNA extracted arabadopsis and <i> Picea abies </i> to improve yield and quality. Carene still failed to get an RNA concentration greater than 10 ng/ul, while Arabidopsis produced 107 ng/ul with a good A260/A280 ratio. <br />
</li><br />
</ul><br />
<br />
<p><b>September 20<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Miniprep of K546546 liquid cultures (1 ml). First culture tube concentration of 85.7 ng/ul DNA, second 105.1 ng/ul<br />
</li><br />
</ul><br />
<br />
<p><b>September 26<sup>th</sup></b></p><br />
<ul><br />
<li><br />
RT-PCR done on humelene, linalool (S), sabinene, and zingiberene. Sabinene produces clear bands, while zingiberene shows one faint band at roughly the correct size. Positive controls are also run to confirm that the reverse transcription step is not the cause of any failures to amplify. <br />
</li><br />
</ul><br />
<br />
<p><b>October 5<sup>th</sup></b></p><br />
<ul><br />
<li><br />
RT-PCR done on humelene, sabinene, and santalene. Sabinene again produces clear bands,and santalene does as well although much <br />
fainter. Both bands were gel extracted to yield a small (<10 ng/ul) amount of DNA.<br />
</li><br />
<li><br />
Extracted DNA was ligated into pUC19 and transformed into E. coli.<br />
</li><br />
</ul><br />
<br />
<p><b>October 7<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Site direction mutagenesis kit and specially designed primers were used to mutagenize K546546 at its BglI site, sabinene cDNA at <br />
its XbaI and EcoRI sites, and pVU14004 at its EcoRI and XbaI sites. <br />
</li><br />
<li><br />
Mutagenesis product was transformed into E. coli.<br />
</li><br />
</ul><br />
<br />
<p><b>October 9<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Minipreps were done on 5 ml liquid cultures of mutagenized pVU14004, sabinene, and K546546. 4 liquid cultures were made for each, and both sabinene and pVU14004 were done in duplicate. <br />
</li><br />
<li><br />
Diagnostic digests were done on miniprepped plasmid to check if the restriction sites were mutagenized. pVU14004 appeared to have lost its XbaI site but not its EcoRI site, sabinene shows a size that suggests it failed to ligate as an insert into pUC19, and K546 shows only a single band at around its starting weight.<br />
</li><br />
</ul><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/d/d9/VU_Diagnostic_Digest_10-9.JPG" align="right" width="320"><br />
<br />
<p><b>October 10<sup>th</sup></b></p><br />
<ul><br />
<li><br />
All 8 minipreps of sabinene ligated into pUC19 were digested with SpeI and ApaI to check for the synthase insert. Only one, Sab B2, shows a second band at the right size.<br />
</li><br />
</ul><br />
<br />
<p><b>October 11<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Santalene synthase was ligated into pVU14004 and pSB1C3. E. coli was transformed and incubated.<br />
</li><br />
<li><br />
The sites that failed to show mutagenesis were mutagenized again using and transformed into E. coli. K546546 had its AgeI site mutagenized. <br />
</li><br />
</ul><br />
<br />
<p><b>October 12<sup>th</sup></b></p><br />
<ul><br />
<li><br />
All liquid cultures were miniprepped, producing 8 samples of pVU14004 with confirmed XbaI mutagenesis, 4 pVU14004 with no sites confirmed, 4 sabinene, and 6 samples produced from santalene in pVU14004. None of the plates with santalene in pSB1C3 produced colonies.<br />
</li><br />
<li><br />
Diagnostic digests were run on all miniprepped plasmid (K546- AgeI, BglI, SphI. Sabinene- EcoRI, BamH1. Santalene in pVU- ApaI, XbaI. pVU- EcoRI, XbaI, BamHI, KpnI). Santalene appeared not to have ligated into pVU14004. Sabinene did not have its EcoRI site removed by mutagenesis. K546546 had at least one cut, but the second sample may have had one site mutagenized. <br />
</li><br />
<li><br />
Santalene was re-ligated into pSB1C3 and transformed. <br />
</li><br />
</ul><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/5/53/VU_10-11_diagnostic_digest.JPG" align="right" width="250" ><br />
<br />
<p><b>October 13<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Santalene in pSB1C3 was miniprepped to good yield. Each of 4 replicates was digested with SpeI and ApaI to test for ligation. The ApaI enzyme appears not have cut, but the fourth sample showed an uncut plasmid size which corresponded to that of pSB1C with santalene successfully inserted. <br />
</li><br />
</ul><br />
<br />
<br />
<p><b>October 14<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Ligated santalene synthase again into pVU14004 and transformed into E. coli<br />
</li><br />
</ul><br />
<br />
<p><b>October 15<sup>th</sup></b></p><br />
<ul><br />
<li><br />
One colony grew and was put in liquid culture. <br />
</li><br />
<li><br />
Culture miniprepped and digested. Again no gene insertion was detectable.<br />
</li><br />
</ul><br />
<br />
<p><b>October 16<sup>th</sup></b></p><br />
<ul><br />
<li><br />
Transformed pVU14004 into a dam- strain of E. coli to address the methylation sensitivity of ApaI. <br />
</li><br />
<li><br />
Finished planning and acquiring materials for GC-MS confirmation of terpene presence.<br />
</li><br />
</ul><br />
<br><br />
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<h3> BBa_K1322231- Optimized Santalene Synthase</h3><br />
<p><br />
BBa_K1322231 is a codon-optimized biobrick part encoding the gene for alpha-santalene synthase (EC 4.2.3.82). The enzyme catalyze the conversion of the common isoprenoid intermediate farnesyl pyrophosphate (FPP) into the sesquiterpene (+)-alpha-santelene in a single step. Traces of (-)-beta-santalene and bergamontene have previously been shown to be produced by this enzyme as well.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/0/0b/VU_San_psb_confirmation.jpg" align="left" width="340" style="padding-bottom:0.5em; float:left" /><br />
<br />
<p><br />
The gene, derived from a relative of the exotic sandalwood tree, has been demonstrated to produce functional terpene product in both yeast (Scalcinati et al 2012) and E. coli (data pending). This is possible due to several endogenous pathways that produce FPP as an intermediate, including the MEV and MEP pathways.<br />
</p><br />
<br />
<p><br />
In addition to being a prized fragrance, with what is often described as a warm, sweet woody scent, the sandalwood oil has been investigated for a number of other practical applications, including as a chemoprotective against carcinogenesis (Banaerjee, Ecavade, and Rao 1993) and inhibitors of viral reproduction (Koch et al 2008).<br />
</p><br />
<br />
<p><br />
Our biobrick has additional functionality added to it beyond just the coding sequence for santalene synthase. Immediately before the start codon is a yeast consensus sequence to permit efficient translation of the gene transcript in <i> S. cerevisiae</i>. Toward the end of the sequence there is also a sequence added inside the reading frame that encodes for a strep tag. The strep tag is a small, eight amino acid epitope tag that is translated onto the C terminus of the recombinant polypeptide. Its small size ensures that it will not likely interfere with protein function, yet in most situations it is still prominent enough that the common molecule streptavidin (in the form of Strep-tactin) can recognize and bind to it. Because anti-streptavidin antibodies are widely available, this opens the way for a range of possibilities, including simple confirmation assays of synthase expression by western blotting and quick purification of the synthase enzyme.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/5/57/VU_pVU14006.png" align="right" alt="pVU14006" width="600" style="padding-bottom:0.5em; float:right" /><br />
<br />
<p><br />
We confirmed our part succesfully ligated into pSB1C3 by a diagnostic digest of miniprepped plasmid. While none of the samples had the santalene insert cleanly cut out, two observations were made which confirmed that in the fourth miniprepped sample, santalene had been successfully integrated. First, the size of the uncut fourth plasmid is equal to pSB1C3 plus the santalene synthase gene. Second, it was later noticed that the plasmids were grown in a non-demethylated strain of E. coli. This would explain why no inserts were cut out, since one enzyme used, ApaI, was dam methylation sensitive. This would also explain why the digested product is consistently larger than the undigested, since the linearized DNA should be above the uncut supercoiled form (Tirabassi 2014). <br />
</p><br />
<br />
<h3> pVU14006 </h3><br />
<p><br />
As a shuttle vector, pVU14006 is capable of expression both in E. coli and S. cerevisiae. It has resistance markers to both ampicillin and kanomycin, making selection convenient in both bacteria and yeast. For cloning in bacteria, it has a prokaryotic origin of replication taken out of pUC19. Two regions of base pair homology with the S. cerevisiae genome allow it to efficiently integrate into the yeast genome. Genomic integration has a number of advantages, including the potential for increased product yield. There is a multiple cloning site with a range of different restriction enzymes to make the plasmid compatible with almost all of the most commonly used restriction enzymes, including those used in RFC10 compatible biobricks. <br />
</p><br />
<br />
<p><br />
A Gal1 inducible promoter is upstream of where the protein coding gene would be inserted. This promoter is strongly repressed by glucose and further allows the protein coding gene to be transcriptionally up-regulated upon the addition of galactose. Changing which of these two carbohydrates are present in the growth media therefore gives an enormous degree of control over the level of gene expression. Finally, a CYC1 terminator is present to ensure proper termination of transcription. <br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/9/9c/Sabinene_synthesis_pathway.gif" align="left" width="500" style="padding-bottom:0.5em; float:right" /><br />
<h3> BBa_K1322001- All-RFC Compatible Fluorescent Oscillator</h3><br />
<br />
<p><br />
As part of our collaboration with Vanderbilt Microfluidics, we were working with the existing biobrick K546546, which encodes a self-regulating fluorescent oscillating system. We wanted to make this part compatible with all major RFC standards, and did so by using our site-directed mutagenesis kit with specially designed primers. The sequence changes were designed so that the part should function equally well as it did before. For a gel showing confirmation, please see our lab notebook under October 11th. <br />
</p><br />
<br />
<h3> Sabinene Synthase </h3><br />
<p><br />
Although it is not yet RFC10 compatible and thus will not appear in the registry until later, we have successfully extracted the gene for sabinene synthase out of raw plant RNA. Following successful mutagenesis, we plan to add the biobrick prefix and suffixes so that this part can be made available at the registry for other iGEM teams to use.<br />
</p><br />
<br />
<br><br />
<h3>References:</h3><br />
<p><i>Scalcinati et al.: Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae. Microbial Cell Factories 2012 11:117<br><br />
Banerjee, Ecavade and Rao: Modulatory influence of sandalwood oil on mouse hepatic glutathione S-transferase activity and acid soluble sulphydryl level. Cancer Letters, 68 (1993) 105 - 109 <br />
Koch et al: Inhibitory effect of essential oils against herpes simplex virus type 2. Phytomedicine 2008;15(1-2):71-8. <br><br />
Rebecca Tirabassi, How to identify supercoils, nicks and circles in plasmid preps. Bitesizebio. October 8, 2014.</i><p></div>Hwangashttp://2014.igem.org/Team:Vanderbilt/PartsTeam:Vanderbilt/Parts2015-02-08T21:17:06Z<p>Hwangas: </p>
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<h3> BBa_K1322231- Optimized Santalene Synthase</h3><br />
<p><br />
BBa_K1322231 is a codon-optimized biobrick part encoding the gene for alpha-santalene synthase (EC 4.2.3.82). The enzyme catalyze the conversion of the common isoprenoid intermediate farnesyl pyrophosphate (FPP) into the sesquiterpene (+)-alpha-santelene in a single step. Traces of (-)-beta-santalene and bergamontene have previously been shown to be produced by this enzyme as well.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/0/0b/VU_San_psb_confirmation.jpg" align="left" width="340" style="padding-bottom:0.5em; float:left" /><br />
<br />
<p><br />
The gene, derived from a relative of the exotic sandalwood tree, has been demonstrated to produce functional terpene product in both yeast (Scalcinati et al 2012) and E. coli (data pending). This is possible due to several endogenous pathways that produce FPP as an intermediate, including the MEV and MEP pathways.<br />
</p><br />
<br />
<p><br />
In addition to being a prized fragrance, with what is often described as a warm, sweet woody scent, the sandalwood oil has been investigated for a number of other practical applications, including as a chemoprotective against carcinogenesis (Banaerjee, Ecavade, and Rao 1993) and inhibitors of viral reproduction (Koch et al 2008).<br />
</p><br />
<br />
<p><br />
Our biobrick has additional functionality added to it beyond just the coding sequence for santalene synthase. Immediately before the start codon is a yeast consensus sequence to permit efficient translation of the gene transcript in <i> S. cerevisiae</i>. Toward the end of the sequence there is also a sequence added inside the reading frame that encodes for a strep tag. The strep tag is a small, eight amino acid epitope tag that is translated onto the C terminus of the recombinant polypeptide. Its small size ensures that it will not likely interfere with protein function, yet in most situations it is still prominent enough that the common molecule streptavidin (in the form of Strep-tactin) can recognize and bind to it. Because anti-streptavidin antibodies are widely available, this opens the way for a range of possibilities, including simple confirmation assays of synthase expression by western blotting and quick purification of the synthase enzyme.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/5/57/VU_pVU14006.png" align="right" alt="pVU14006" width="600" style="padding-bottom:0.5em; float:right" /><br />
<br />
<p><br />
We confirmed our part succesfully ligated into pSB1C3 by a diagnostic digest of miniprepped plasmid. While none of the samples had the santalene insert cleanly cut out, two observations were made which confirmed that in the fourth miniprepped sample, santalene had been successfully integrated. First, the size of the uncut fourth plasmid is equal to pSB1C3 plus the santalene synthase gene. Second, it was later noticed that the plasmids were grown in a non-demethylated strain of E. coli. This would explain why no inserts were cut out, since one enzyme used, ApaI, was dam methylation sensitive. This would also explain why the digested product is consistently larger than the undigested, since the linearized DNA should be above the uncut supercoiled form (Tirabassi 2014). <br />
</p><br />
<br />
<h3> pVU14006 </h3><br />
<p><br />
As a shuttle vector, pVU14006 is capable of expression both in E. coli and S. cerevisiae. It has resistance markers to both ampicillin and kanomycin, making selection convenient in both bacteria and yeast. For cloning in bacteria, it has a prokaryotic origin of replication taken out of pUC19. Two regions of base pair homology with the S. cerevisiae genome allow it to efficiently integrate into the yeast genome. Genomic integration has a number of advantages, including the potential for increased product yield. There is a multiple cloning site with a range of different restriction enzymes to make the plasmid compatible with almost all of the most commonly used restriction enzymes, including those used in RFC10 compatible biobricks. <br />
</p><br />
<br />
<p><br />
A Gal1 inducible promoter is upstream of where the protein coding gene would be inserted. This promoter is strongly repressed by glucose and further allows the protein coding gene to be transcriptionally up-regulated upon the addition of galactose. Changing which of these two carbohydrates are present in the growth media therefore gives an enormous degree of control over the level of gene expression. Finally, a CYC1 terminator is present to ensure proper termination of transcription. <br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/9/9c/Sabinene_synthesis_pathway.gif" align="left" width="500" style="padding-bottom:0.5em; float:left" /><br />
<h3> BBa_K1322001- All-RFC Compatible Fluorescent Oscillator</h3><br />
<br />
<p><br />
As part of our collaboration with Vanderbilt Microfluidics, we were working with the existing biobrick K546546, which encodes a self-regulating fluorescent oscillating system. We wanted to make this part compatible with all major RFC standards, and did so by using our site-directed mutagenesis kit with specially designed primers. The sequence changes were designed so that the part should function equally well as it did before. For a gel showing confirmation, please see our lab notebook under October 11th. <br />
</p><br />
<br />
<h3> Sabinene Synthase </h3><br />
<p><br />
Although it is not yet RFC10 compatible and thus will not appear in the registry until later, we have successfully extracted the gene for sabinene synthase out of raw plant RNA. Following successful mutagenesis, we plan to add the biobrick prefix and suffixes so that this part can be made available at the registry for other iGEM teams to use.<br />
</p><br />
<br />
<br><br />
<h3>References:</h3><br />
<p><i>Scalcinati et al.: Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae. Microbial Cell Factories 2012 11:117<br><br />
Banerjee, Ecavade and Rao: Modulatory influence of sandalwood oil on mouse hepatic glutathione S-transferase activity and acid soluble sulphydryl level. Cancer Letters, 68 (1993) 105 - 109 <br />
Koch et al: Inhibitory effect of essential oils against herpes simplex virus type 2. Phytomedicine 2008;15(1-2):71-8. <br><br />
Rebecca Tirabassi, How to identify supercoils, nicks and circles in plasmid preps. Bitesizebio. October 8, 2014.</i><p></div>Hwangashttp://2014.igem.org/Team:Vanderbilt/PartsTeam:Vanderbilt/Parts2015-02-08T21:16:50Z<p>Hwangas: </p>
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background-repeat: no-repeat;<br />
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font-family: Georgia, Times, "Times New Roman", serif; <br />
}<br />
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table {<br />
cellpadding: 10;<br />
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<h3> BBa_K1322231- Optimized Santalene Synthase</h3><br />
<p><br />
BBa_K1322231 is a codon-optimized biobrick part encoding the gene for alpha-santalene synthase (EC 4.2.3.82). The enzyme catalyze the conversion of the common isoprenoid intermediate farnesyl pyrophosphate (FPP) into the sesquiterpene (+)-alpha-santelene in a single step. Traces of (-)-beta-santalene and bergamontene have previously been shown to be produced by this enzyme as well.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/0/0b/VU_San_psb_confirmation.jpg" align="left" width="340" style="padding-bottom:0.5em; float:left" /><br />
<br />
<p><br />
The gene, derived from a relative of the exotic sandalwood tree, has been demonstrated to produce functional terpene product in both yeast (Scalcinati et al 2012) and E. coli (data pending). This is possible due to several endogenous pathways that produce FPP as an intermediate, including the MEV and MEP pathways.<br />
</p><br />
<br />
<p><br />
In addition to being a prized fragrance, with what is often described as a warm, sweet woody scent, the sandalwood oil has been investigated for a number of other practical applications, including as a chemoprotective against carcinogenesis (Banaerjee, Ecavade, and Rao 1993) and inhibitors of viral reproduction (Koch et al 2008).<br />
</p><br />
<br />
<p><br />
Our biobrick has additional functionality added to it beyond just the coding sequence for santalene synthase. Immediately before the start codon is a yeast consensus sequence to permit efficient translation of the gene transcript in <i> S. cerevisiae</i>. Toward the end of the sequence there is also a sequence added inside the reading frame that encodes for a strep tag. The strep tag is a small, eight amino acid epitope tag that is translated onto the C terminus of the recombinant polypeptide. Its small size ensures that it will not likely interfere with protein function, yet in most situations it is still prominent enough that the common molecule streptavidin (in the form of Strep-tactin) can recognize and bind to it. Because anti-streptavidin antibodies are widely available, this opens the way for a range of possibilities, including simple confirmation assays of synthase expression by western blotting and quick purification of the synthase enzyme.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/5/57/VU_pVU14006.png" align="right" alt="pVU14006" width="600" style="padding-bottom:0.5em; float:right" /><br />
<br />
<p><br />
We confirmed our part succesfully ligated into pSB1C3 by a diagnostic digest of miniprepped plasmid. While none of the samples had the santalene insert cleanly cut out, two observations were made which confirmed that in the fourth miniprepped sample, santalene had been successfully integrated. First, the size of the uncut fourth plasmid is equal to pSB1C3 plus the santalene synthase gene. Second, it was later noticed that the plasmids were grown in a non-demethylated strain of E. coli. This would explain why no inserts were cut out, since one enzyme used, ApaI, was dam methylation sensitive. This would also explain why the digested product is consistently larger than the undigested, since the linearized DNA should be above the uncut supercoiled form (Tirabassi 2014). <br />
</p><br />
<br />
<h3> pVU14006 </h3><br />
<p><br />
As a shuttle vector, pVU14006 is capable of expression both in E. coli and S. cerevisiae. It has resistance markers to both ampicillin and kanomycin, making selection convenient in both bacteria and yeast. For cloning in bacteria, it has a prokaryotic origin of replication taken out of pUC19. Two regions of base pair homology with the S. cerevisiae genome allow it to efficiently integrate into the yeast genome. Genomic integration has a number of advantages, including the potential for increased product yield. There is a multiple cloning site with a range of different restriction enzymes to make the plasmid compatible with almost all of the most commonly used restriction enzymes, including those used in RFC10 compatible biobricks. <br />
</p><br />
<br />
<p><br />
A Gal1 inducible promoter is upstream of where the protein coding gene would be inserted. This promoter is strongly repressed by glucose and further allows the protein coding gene to be transcriptionally up-regulated upon the addition of galactose. Changing which of these two carbohydrates are present in the growth media therefore gives an enormous degree of control over the level of gene expression. Finally, a CYC1 terminator is present to ensure proper termination of transcription. <br />
</p><br />
<br />
<h3> BBa_K1322001- All-RFC Compatible Fluorescent Oscillator</h3><br />
<img src="https://static.igem.org/mediawiki/parts/9/9c/Sabinene_synthesis_pathway.gif" align="left" width="500" style="padding-bottom:0.5em; float:left" /><br />
<br />
<p><br />
As part of our collaboration with Vanderbilt Microfluidics, we were working with the existing biobrick K546546, which encodes a self-regulating fluorescent oscillating system. We wanted to make this part compatible with all major RFC standards, and did so by using our site-directed mutagenesis kit with specially designed primers. The sequence changes were designed so that the part should function equally well as it did before. For a gel showing confirmation, please see our lab notebook under October 11th. <br />
</p><br />
<br />
<h3> Sabinene Synthase </h3><br />
<p><br />
Although it is not yet RFC10 compatible and thus will not appear in the registry until later, we have successfully extracted the gene for sabinene synthase out of raw plant RNA. Following successful mutagenesis, we plan to add the biobrick prefix and suffixes so that this part can be made available at the registry for other iGEM teams to use.<br />
</p><br />
<br />
<br><br />
<h3>References:</h3><br />
<p><i>Scalcinati et al.: Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae. Microbial Cell Factories 2012 11:117<br><br />
Banerjee, Ecavade and Rao: Modulatory influence of sandalwood oil on mouse hepatic glutathione S-transferase activity and acid soluble sulphydryl level. Cancer Letters, 68 (1993) 105 - 109 <br />
Koch et al: Inhibitory effect of essential oils against herpes simplex virus type 2. Phytomedicine 2008;15(1-2):71-8. <br><br />
Rebecca Tirabassi, How to identify supercoils, nicks and circles in plasmid preps. Bitesizebio. October 8, 2014.</i><p></div>Hwangashttp://2014.igem.org/Team:Vanderbilt/PartsTeam:Vanderbilt/Parts2015-02-08T21:16:16Z<p>Hwangas: </p>
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<img src="https://static.igem.org/mediawiki/parts/f/f4/VU_Santalene_biosynthesis_path.gif" align=right width="500" style="padding-bottom:0.5em;" /><br />
<h3> BBa_K1322231- Optimized Santalene Synthase</h3><br />
<p><br />
BBa_K1322231 is a codon-optimized biobrick part encoding the gene for alpha-santalene synthase (EC 4.2.3.82). The enzyme catalyze the conversion of the common isoprenoid intermediate farnesyl pyrophosphate (FPP) into the sesquiterpene (+)-alpha-santelene in a single step. Traces of (-)-beta-santalene and bergamontene have previously been shown to be produced by this enzyme as well.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/0/0b/VU_San_psb_confirmation.jpg" align="left" width="340" style="padding-bottom:0.5em; float:left" /><br />
<br />
<p><br />
The gene, derived from a relative of the exotic sandalwood tree, has been demonstrated to produce functional terpene product in both yeast (Scalcinati et al 2012) and E. coli (data pending). This is possible due to several endogenous pathways that produce FPP as an intermediate, including the MEV and MEP pathways.<br />
</p><br />
<br />
<p><br />
In addition to being a prized fragrance, with what is often described as a warm, sweet woody scent, the sandalwood oil has been investigated for a number of other practical applications, including as a chemoprotective against carcinogenesis (Banaerjee, Ecavade, and Rao 1993) and inhibitors of viral reproduction (Koch et al 2008).<br />
</p><br />
<br />
<p><br />
Our biobrick has additional functionality added to it beyond just the coding sequence for santalene synthase. Immediately before the start codon is a yeast consensus sequence to permit efficient translation of the gene transcript in <i> S. cerevisiae</i>. Toward the end of the sequence there is also a sequence added inside the reading frame that encodes for a strep tag. The strep tag is a small, eight amino acid epitope tag that is translated onto the C terminus of the recombinant polypeptide. Its small size ensures that it will not likely interfere with protein function, yet in most situations it is still prominent enough that the common molecule streptavidin (in the form of Strep-tactin) can recognize and bind to it. Because anti-streptavidin antibodies are widely available, this opens the way for a range of possibilities, including simple confirmation assays of synthase expression by western blotting and quick purification of the synthase enzyme.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/5/57/VU_pVU14006.png" align="right" alt="pVU14006" width="600" style="padding-bottom:0.5em; float:right" /><br />
<br />
<p><br />
We confirmed our part succesfully ligated into pSB1C3 by a diagnostic digest of miniprepped plasmid. While none of the samples had the santalene insert cleanly cut out, two observations were made which confirmed that in the fourth miniprepped sample, santalene had been successfully integrated. First, the size of the uncut fourth plasmid is equal to pSB1C3 plus the santalene synthase gene. Second, it was later noticed that the plasmids were grown in a non-demethylated strain of E. coli. This would explain why no inserts were cut out, since one enzyme used, ApaI, was dam methylation sensitive. This would also explain why the digested product is consistently larger than the undigested, since the linearized DNA should be above the uncut supercoiled form (Tirabassi 2014). <br />
</p><br />
<br />
<h3> pVU14006 </h3><br />
<p><br />
As a shuttle vector, pVU14006 is capable of expression both in E. coli and S. cerevisiae. It has resistance markers to both ampicillin and kanomycin, making selection convenient in both bacteria and yeast. For cloning in bacteria, it has a prokaryotic origin of replication taken out of pUC19. Two regions of base pair homology with the S. cerevisiae genome allow it to efficiently integrate into the yeast genome. Genomic integration has a number of advantages, including the potential for increased product yield. There is a multiple cloning site with a range of different restriction enzymes to make the plasmid compatible with almost all of the most commonly used restriction enzymes, including those used in RFC10 compatible biobricks. <br />
</p><br />
<br />
<p><br />
A Gal1 inducible promoter is upstream of where the protein coding gene would be inserted. This promoter is strongly repressed by glucose and further allows the protein coding gene to be transcriptionally up-regulated upon the addition of galactose. Changing which of these two carbohydrates are present in the growth media therefore gives an enormous degree of control over the level of gene expression. Finally, a CYC1 terminator is present to ensure proper termination of transcription. <br />
</p><br />
<br />
<h3> BBa_K1322001- All-RFC Compatible Fluorescent Oscillator</h3><br />
<img src="https://static.igem.org/mediawiki/parts/9/9c/Sabinene_synthesis_pathway.gif" align="left" width="500" style="padding-bottom:0.5em; float:left" /><br />
<br />
<p><br />
As part of our collaboration with Vanderbilt Microfluidics, we were working with the existing biobrick K546546, which encodes a self-regulating fluorescent oscillating system. We wanted to make this part compatible with all major RFC standards, and did so by using our site-directed mutagenesis kit with specially designed primers. The sequence changes were designed so that the part should function equally well as it did before. For a gel showing confirmation, please see our lab notebook under October 11th. <br />
</p><br />
<br />
<h3> Sabinene Synthase </h3><br />
<p><br />
Although it is not yet RFC10 compatible and thus will not appear in the registry until later, we have successfully extracted the gene for sabinene synthase out of raw plant RNA. Following successful mutagenesis, we plan to add the biobrick prefix and suffixes so that this part can be made available at the registry for other iGEM teams to use.<br />
</p><br />
<br />
<br><br />
<h3>References:</h3><br />
<p><i>Scalcinati et al.: Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae. Microbial Cell Factories 2012 11:117<br><br />
Banerjee, Ecavade and Rao: Modulatory influence of sandalwood oil on mouse hepatic glutathione S-transferase activity and acid soluble sulphydryl level. Cancer Letters, 68 (1993) 105 - 109 <br />
Koch et al: Inhibitory effect of essential oils against herpes simplex virus type 2. Phytomedicine 2008;15(1-2):71-8. <br><br />
Rebecca Tirabassi, How to identify supercoils, nicks and circles in plasmid preps. Bitesizebio. October 8, 2014.</i><p></div>Hwangashttp://2014.igem.org/Team:Vanderbilt/PartsTeam:Vanderbilt/Parts2015-02-08T21:15:48Z<p>Hwangas: </p>
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font-family: Georgia, Times, "Times New Roman", serif; <br />
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<h3> BBa_K1322231- Optimized Santalene Synthase</h3><br />
<p><br />
BBa_K1322231 is a codon-optimized biobrick part encoding the gene for alpha-santalene synthase (EC 4.2.3.82). The enzyme catalyze the conversion of the common isoprenoid intermediate farnesyl pyrophosphate (FPP) into the sesquiterpene (+)-alpha-santelene in a single step. Traces of (-)-beta-santalene and bergamontene have previously been shown to be produced by this enzyme as well.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/0/0b/VU_San_psb_confirmation.jpg" align="left" width="340" style="padding-bottom:0.5em; float:left" /><br />
<br />
<p><br />
The gene, derived from a relative of the exotic sandalwood tree, has been demonstrated to produce functional terpene product in both yeast (Scalcinati et al 2012) and E. coli (data pending). This is possible due to several endogenous pathways that produce FPP as an intermediate, including the MEV and MEP pathways.<br />
</p><br />
<br />
<p><br />
In addition to being a prized fragrance, with what is often described as a warm, sweet woody scent, the sandalwood oil has been investigated for a number of other practical applications, including as a chemoprotective against carcinogenesis (Banaerjee, Ecavade, and Rao 1993) and inhibitors of viral reproduction (Koch et al 2008).<br />
</p><br />
<br />
<p><br />
Our biobrick has additional functionality added to it beyond just the coding sequence for santalene synthase. Immediately before the start codon is a yeast consensus sequence to permit efficient translation of the gene transcript in <i> S. cerevisiae</i>. Toward the end of the sequence there is also a sequence added inside the reading frame that encodes for a strep tag. The strep tag is a small, eight amino acid epitope tag that is translated onto the C terminus of the recombinant polypeptide. Its small size ensures that it will not likely interfere with protein function, yet in most situations it is still prominent enough that the common molecule streptavidin (in the form of Strep-tactin) can recognize and bind to it. Because anti-streptavidin antibodies are widely available, this opens the way for a range of possibilities, including simple confirmation assays of synthase expression by western blotting and quick purification of the synthase enzyme.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/5/57/VU_pVU14006.png" align="right" alt="pVU14006" width="600" style="padding-bottom:0.5em; float:right" /><br />
<br />
<p><br />
We confirmed our part succesfully ligated into pSB1C3 by a diagnostic digest of miniprepped plasmid. While none of the samples had the santalene insert cleanly cut out, two observations were made which confirmed that in the fourth miniprepped sample, santalene had been successfully integrated. First, the size of the uncut fourth plasmid is equal to pSB1C3 plus the santalene synthase gene. Second, it was later noticed that the plasmids were grown in a non-demethylated strain of E. coli. This would explain why no inserts were cut out, since one enzyme used, ApaI, was dam methylation sensitive. This would also explain why the digested product is consistently larger than the undigested, since the linearized DNA should be above the uncut supercoiled form (Tirabassi 2014). <br />
</p><br />
<br />
<h3> pVU14006 </h3><br />
<p><br />
As a shuttle vector, pVU14006 is capable of expression both in E. coli and S. cerevisiae. It has resistance markers to both ampicillin and kanomycin, making selection convenient in both bacteria and yeast. For cloning in bacteria, it has a prokaryotic origin of replication taken out of pUC19. Two regions of base pair homology with the S. cerevisiae genome allow it to efficiently integrate into the yeast genome. Genomic integration has a number of advantages, including the potential for increased product yield. There is a multiple cloning site with a range of different restriction enzymes to make the plasmid compatible with almost all of the most commonly used restriction enzymes, including those used in RFC10 compatible biobricks. <br />
</p><br />
<br />
<p><br />
A Gal1 inducible promoter is upstream of where the protein coding gene would be inserted. This promoter is strongly repressed by glucose and further allows the protein coding gene to be transcriptionally up-regulated upon the addition of galactose. Changing which of these two carbohydrates are present in the growth media therefore gives an enormous degree of control over the level of gene expression. Finally, a CYC1 terminator is present to ensure proper termination of transcription. <br />
</p><br />
<br />
<h3> BBa_K1322001- All-RFC Compatible Fluorescent Oscillator</h3><br />
<img src="https://static.igem.org/mediawiki/parts/9/9c/Sabinene_synthesis_pathway.gif" align="left" width="500" style="padding-bottom:0.5em; float:left" /><br />
<br />
<p><br />
As part of our collaboration with Vanderbilt Microfluidics, we were working with the existing biobrick K546546, which encodes a self-regulating fluorescent oscillating system. We wanted to make this part compatible with all major RFC standards, and did so by using our site-directed mutagenesis kit with specially designed primers. The sequence changes were designed so that the part should function equally well as it did before. For a gel showing confirmation, please see our lab notebook under October 11th. <br />
</p><br />
<br />
<h3> Sabinene Synthase </h3><br />
<p><br />
Although it is not yet RFC10 compatible and thus will not appear in the registry until later, we have successfully extracted the gene for sabinene synthase out of raw plant RNA. Following successful mutagenesis, we plan to add the biobrick prefix and suffixes so that this part can be made available at the registry for other iGEM teams to use.<br />
</p><br />
<br />
<br><br />
<h3>References:</h3><br />
<p><i>Scalcinati et al.: Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae. Microbial Cell Factories 2012 11:117<br><br />
Banerjee, Ecavade and Rao: Modulatory influence of sandalwood oil on mouse hepatic glutathione S-transferase activity and acid soluble sulphydryl level. Cancer Letters, 68 (1993) 105 - 109 <br />
Koch et al: Inhibitory effect of essential oils against herpes simplex virus type 2. Phytomedicine 2008;15(1-2):71-8. <br><br />
Rebecca Tirabassi, How to identify supercoils, nicks and circles in plasmid preps. Bitesizebio. October 8, 2014.</i><p></div>Hwangashttp://2014.igem.org/Team:Vanderbilt/PartsTeam:Vanderbilt/Parts2015-02-08T21:14:59Z<p>Hwangas: </p>
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<img src="https://static.igem.org/mediawiki/parts/f/f4/VU_Santalene_biosynthesis_path.gif" align="right" width="500" style="padding-bottom:0.5em; float:right" /><br />
<h3> BBa_K1322231- Optimized Santalene Synthase</h3><br />
<p><br />
BBa_K1322231 is a codon-optimized biobrick part encoding the gene for alpha-santalene synthase (EC 4.2.3.82). The enzyme catalyze the conversion of the common isoprenoid intermediate farnesyl pyrophosphate (FPP) into the sesquiterpene (+)-alpha-santelene in a single step. Traces of (-)-beta-santalene and bergamontene have previously been shown to be produced by this enzyme as well.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/0/0b/VU_San_psb_confirmation.jpg" align="left" width="340" style="padding-bottom:0.5em; float:left" /><br />
<br />
<p><br />
The gene, derived from a relative of the exotic sandalwood tree, has been demonstrated to produce functional terpene product in both yeast (Scalcinati et al 2012) and E. coli (data pending). This is possible due to several endogenous pathways that produce FPP as an intermediate, including the MEV and MEP pathways.<br />
</p><br />
<br />
<p><br />
In addition to being a prized fragrance, with what is often described as a warm, sweet woody scent, the sandalwood oil has been investigated for a number of other practical applications, including as a chemoprotective against carcinogenesis (Banaerjee, Ecavade, and Rao 1993) and inhibitors of viral reproduction (Koch et al 2008).<br />
</p><br />
<br />
<p><br />
Our biobrick has additional functionality added to it beyond just the coding sequence for santalene synthase. Immediately before the start codon is a yeast consensus sequence to permit efficient translation of the gene transcript in <i> S. cerevisiae</i>. Toward the end of the sequence there is also a sequence added inside the reading frame that encodes for a strep tag. The strep tag is a small, eight amino acid epitope tag that is translated onto the C terminus of the recombinant polypeptide. Its small size ensures that it will not likely interfere with protein function, yet in most situations it is still prominent enough that the common molecule streptavidin (in the form of Strep-tactin) can recognize and bind to it. Because anti-streptavidin antibodies are widely available, this opens the way for a range of possibilities, including simple confirmation assays of synthase expression by western blotting and quick purification of the synthase enzyme.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/5/57/VU_pVU14006.png" align="right" alt="pVU14006" width="600" style="padding-bottom:0.5em; float:right" /><br />
<br />
<p><br />
We confirmed our part succesfully ligated into pSB1C3 by a diagnostic digest of miniprepped plasmid. While none of the samples had the santalene insert cleanly cut out, two observations were made which confirmed that in the fourth miniprepped sample, santalene had been successfully integrated. First, the size of the uncut fourth plasmid is equal to pSB1C3 plus the santalene synthase gene. Second, it was later noticed that the plasmids were grown in a non-demethylated strain of E. coli. This would explain why no inserts were cut out, since one enzyme used, ApaI, was dam methylation sensitive. This would also explain why the digested product is consistently larger than the undigested, since the linearized DNA should be above the uncut supercoiled form (Tirabassi 2014). <br />
</p><br />
<br />
<h3> pVU14006 </h3><br />
<p><br />
As a shuttle vector, pVU14006 is capable of expression both in E. coli and S. cerevisiae. It has resistance markers to both ampicillin and kanomycin, making selection convenient in both bacteria and yeast. For cloning in bacteria, it has a prokaryotic origin of replication taken out of pUC19. Two regions of base pair homology with the S. cerevisiae genome allow it to efficiently integrate into the yeast genome. Genomic integration has a number of advantages, including the potential for increased product yield. There is a multiple cloning site with a range of different restriction enzymes to make the plasmid compatible with almost all of the most commonly used restriction enzymes, including those used in RFC10 compatible biobricks. <br />
</p><br />
<br />
<p><br />
A Gal1 inducible promoter is upstream of where the protein coding gene would be inserted. This promoter is strongly repressed by glucose and further allows the protein coding gene to be transcriptionally up-regulated upon the addition of galactose. Changing which of these two carbohydrates are present in the growth media therefore gives an enormous degree of control over the level of gene expression. Finally, a CYC1 terminator is present to ensure proper termination of transcription. <br />
</p><br />
<br />
<h3> BBa_K1322001- All-RFC Compatible Fluorescent Oscillator</h3><br />
<img src="https://static.igem.org/mediawiki/parts/9/9c/Sabinene_synthesis_pathway.gif" align="left" width="500" style="padding-bottom:0.5em; float:left" /><br />
<br />
<p><br />
As part of our collaboration with Vanderbilt Microfluidics, we were working with the existing biobrick K546546, which encodes a self-regulating fluorescent oscillating system. We wanted to make this part compatible with all major RFC standards, and did so by using our site-directed mutagenesis kit with specially designed primers. The sequence changes were designed so that the part should function equally well as it did before. For a gel showing confirmation, please see our lab notebook under October 11th. <br />
</p><br />
<br />
<h3> Sabinene Synthase </h3><br />
<br />
Although it is not yet RFC10 compatible and thus will not appear in the registry until later, we have successfully extracted the gene for sabinene synthase out of raw plant RNA. Following successful mutagenesis, we plan to add the biobrick prefix and suffixes so that this part can be made available at the registry for other iGEM teams to use.<br />
<br />
<br><br><br><br><br><br />
<h3>References:</h3><br />
<p><i>Scalcinati et al.: Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae. Microbial Cell Factories 2012 11:117<br><br />
Banerjee, Ecavade and Rao: Modulatory influence of sandalwood oil on mouse hepatic glutathione S-transferase activity and acid soluble sulphydryl level. Cancer Letters, 68 (1993) 105 - 109 <br />
Koch et al: Inhibitory effect of essential oils against herpes simplex virus type 2. Phytomedicine 2008;15(1-2):71-8. <br><br />
Rebecca Tirabassi, How to identify supercoils, nicks and circles in plasmid preps. Bitesizebio. October 8, 2014.</i><p></div>Hwangashttp://2014.igem.org/Team:Vanderbilt/PartsTeam:Vanderbilt/Parts2015-02-08T21:14:22Z<p>Hwangas: </p>
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body {<br />
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background-repeat: no-repeat;<br />
background-attachment: fixed;<br />
background-size:100% auto;<br />
}<br />
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table {<br />
cellpadding: 10;<br />
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margin-left: auto; <br />
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<img src="https://static.igem.org/mediawiki/parts/f/f4/VU_Santalene_biosynthesis_path.gif" align="right" width="500" style="padding-bottom:0.5em; float:right" /><br />
<h3> BBa_K1322231- Optimized Santalene Synthase</h3><br />
<p><br />
BBa_K1322231 is a codon-optimized biobrick part encoding the gene for alpha-santalene synthase (EC 4.2.3.82). The enzyme catalyze the conversion of the common isoprenoid intermediate farnesyl pyrophosphate (FPP) into the sesquiterpene (+)-alpha-santelene in a single step. Traces of (-)-beta-santalene and bergamontene have previously been shown to be produced by this enzyme as well.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/0/0b/VU_San_psb_confirmation.jpg" align="left" width="340" style="padding-bottom:0.5em; float:left" /><br />
<br />
<p><br />
The gene, derived from a relative of the exotic sandalwood tree, has been demonstrated to produce functional terpene product in both yeast (Scalcinati et al 2012) and E. coli (data pending). This is possible due to several endogenous pathways that produce FPP as an intermediate, including the MEV and MEP pathways.<br />
</p><br />
<br />
<p><br />
In addition to being a prized fragrance, with what is often described as a warm, sweet woody scent, the sandalwood oil has been investigated for a number of other practical applications, including as a chemoprotective against carcinogenesis (Banaerjee, Ecavade, and Rao 1993) and inhibitors of viral reproduction (Koch et al 2008).<br />
</p><br />
<br />
<p><br />
Our biobrick has additional functionality added to it beyond just the coding sequence for santalene synthase. Immediately before the start codon is a yeast consensus sequence to permit efficient translation of the gene transcript in <i> S. cerevisiae</i>. Toward the end of the sequence there is also a sequence added inside the reading frame that encodes for a strep tag. The strep tag is a small, eight amino acid epitope tag that is translated onto the C terminus of the recombinant polypeptide. Its small size ensures that it will not likely interfere with protein function, yet in most situations it is still prominent enough that the common molecule streptavidin (in the form of Strep-tactin) can recognize and bind to it. Because anti-streptavidin antibodies are widely available, this opens the way for a range of possibilities, including simple confirmation assays of synthase expression by western blotting and quick purification of the synthase enzyme.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/5/57/VU_pVU14006.png" align="right" alt="pVU14006" width="600" style="padding-bottom:0.5em; float:right" /><br />
<br />
<p><br />
We confirmed our part succesfully ligated into pSB1C3 by a diagnostic digest of miniprepped plasmid. While none of the samples had the santalene insert cleanly cut out, two observations were made which confirmed that in the fourth miniprepped sample, santalene had been successfully integrated. First, the size of the uncut fourth plasmid is equal to pSB1C3 plus the santalene synthase gene. Second, it was later noticed that the plasmids were grown in a non-demethylated strain of E. coli. This would explain why no inserts were cut out, since one enzyme used, ApaI, was dam methylation sensitive. This would also explain why the digested product is consistently larger than the undigested, since the linearized DNA should be above the uncut supercoiled form (Tirabassi 2014). <br />
</p><br />
<br />
<h3> pVU14006 </h3><br />
<p><br />
As a shuttle vector, pVU14006 is capable of expression both in E. coli and S. cerevisiae. It has resistance markers to both ampicillin and kanomycin, making selection convenient in both bacteria and yeast. For cloning in bacteria, it has a prokaryotic origin of replication taken out of pUC19. Two regions of base pair homology with the S. cerevisiae genome allow it to efficiently integrate into the yeast genome. Genomic integration has a number of advantages, including the potential for increased product yield. There is a multiple cloning site with a range of different restriction enzymes to make the plasmid compatible with almost all of the most commonly used restriction enzymes, including those used in RFC10 compatible biobricks. <br />
</p><br />
<br />
<p><br />
A Gal1 inducible promoter is upstream of where the protein coding gene would be inserted. This promoter is strongly repressed by glucose and further allows the protein coding gene to be transcriptionally up-regulated upon the addition of galactose. Changing which of these two carbohydrates are present in the growth media therefore gives an enormous degree of control over the level of gene expression. Finally, a CYC1 terminator is present to ensure proper termination of transcription. <br />
</p><br />
<br />
<h3> BBa_K1322001- All-RFC Compatible Fluorescent Oscillator</h3><br />
<img src="https://static.igem.org/mediawiki/parts/9/9c/Sabinene_synthesis_pathway.gif" align="left" width="500" style="padding-bottom:0.5em; float:left" /><br />
<br />
<p><br />
As part of our collaboration with Vanderbilt Microfluidics, we were working with the existing biobrick K546546, which encodes a self-regulating fluorescent oscillating system. We wanted to make this part compatible with all major RFC standards, and did so by using our site-directed mutagenesis kit with specially designed primers. The sequence changes were designed so that the part should function equally well as it did before. For a gel showing confirmation, please see our lab notebook under October 11th. <br />
</p><br />
<br />
<h3> Sabinene Synthase </h3><br />
<br />
Although it is not yet RFC10 compatible and thus will not appear in the registry until later, we have successfully extracted the gene for sabinene synthase out of raw plant RNA. Following successful mutagenesis, we plan to add the biobrick prefix and suffixes so that this part can be made available at the registry for other iGEM teams to use.<br />
<br />
<br><br><br><br><br><br />
<h3>References:</h3><br />
<p><i>Scalcinati et al.: Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae. Microbial Cell Factories 2012 11:117<br><br />
Banerjee, Ecavade and Rao: Modulatory influence of sandalwood oil on mouse hepatic glutathione S-transferase activity and acid soluble sulphydryl level. Cancer Letters, 68 (1993) 105 - 109 <br />
Koch et al: Inhibitory effect of essential oils against herpes simplex virus type 2. Phytomedicine 2008;15(1-2):71-8. <br><br />
Rebecca Tirabassi, How to identify supercoils, nicks and circles in plasmid preps. Bitesizebio. October 8, 2014.</i><p></div>Hwangashttp://2014.igem.org/Team:Vanderbilt/PartsTeam:Vanderbilt/Parts2015-02-08T21:12:52Z<p>Hwangas: </p>
<hr />
<div>{{CSS/Main}}<br />
<br />
<html><br />
<style type="text/css"><br />
body {<br />
position: relative;<br />
width: 850px //100%;<br />
margin: 0;<br />
padding: 0;<br />
padding-bottom: 10px;<br />
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<h3> BBa_K1322231- Optimized Santalene Synthase</h3><br />
<img src="https://static.igem.org/mediawiki/parts/f/f4/VU_Santalene_biosynthesis_path.gif" align="right" width="500" style="padding-bottom:0.5em; float:right" /><br />
<br />
BBa_K1322231 is a codon-optimized biobrick part encoding the gene for alpha-santalene synthase (EC 4.2.3.82). The enzyme catalyze the conversion of the common isoprenoid intermediate farnesyl pyrophosphate (FPP) into the sesquiterpene (+)-alpha-santelene in a single step. Traces of (-)-beta-santalene and bergamontene have previously been shown to be produced by this enzyme as well.<br />
<br><br><img src="https://static.igem.org/mediawiki/parts/0/0b/VU_San_psb_confirmation.jpg" align="left" width="340" style="padding-bottom:0.5em; float:left" /><br />
The gene, derived from a relative of the exotic sandalwood tree, has been demonstrated to produce functional terpene product in both yeast (Scalcinati et al 2012) and E. coli (data pending). This is possible due to several endogenous pathways that produce FPP as an intermediate, including the MEV and MEP pathways,<br />
<br><br><br />
In addition to being a prized fragrance, with what is often described as a warm, sweet woody scent, the sandalwood oil has been investigated for a number of other practical applications, including as a chemoprotective against carcinogenesis (Banaerjee, Ecavade, and Rao 1993) and inhibitors of viral reproduction (Koch et al 2008).<br />
<br><br><br />
Our biobrick has additional functionality added to it beyond just the coding sequence for santalene synthase. Immediately before the start codon is a yeast consensus sequence to permit efficient translation of the gene transcript in <i> S. cerevisiae</i>. Toward the end of the sequence there is also a sequence added inside the reading frame that encodes for a strep tag. The strep tag is a small, eight amino acid epitope tag that is translated onto the C terminus of the recombinant polypeptide. Its small size ensures that it will not likely interfere with protein function, yet in most situations it is still prominent enough that the common molecule streptavidin (in the form of Strep-tactin) can recognize and bind to it. Because anti-streptavidin antibodies are widely available, this opens the way for a range of possibilities, including simple confirmation assays of synthase expression by western blotting and quick purification of the synthase enzyme.<br />
<br><br><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/5/57/VU_pVU14006.png" align="right" alt="pVU14006" width="600" style="padding-bottom:0.5em; float:right" /><br />
We confirmed our part succesfully ligated into pSB1C3 by a diagnostic digest of miniprepped plasmid. While none of the samples had the santalene insert cleanly cut out, two observations were made which confirmed that in the fourth miniprepped sample, santalene had been successfully integrated. First, the size of the uncut fourth plasmid is equal to pSB1C3 plus the santalene synthase gene. Second, it was later noticed that the plasmids were grown in a non-demethylated strain of E. coli. This would explain why no inserts were cut out, since one enzyme used, ApaI, was dam methylation sensitive. This would also explain why the digested product is consistently larger than the undigested, since the linearized DNA should be above the uncut supercoiled form (Tirabassi 2014). <br />
<br />
<h3> pVU14006 </h3><br />
As a shuttle vector, pVU14006 is capable of expression both in E. coli and S. cerevisiae. It has resistance markers to both ampicillin and kanomycin, making selection convenient in both bacteria and yeast. For cloning in bacteria, it has a prokaryotic origin of replication taken out of pUC19. Two regions of base pair homology with the S. cerevisiae genome allow it to efficiently integrate into the yeast genome. Genomic integration has a number of advantages, including the potential for increased product yield. There is a multiple cloning site with a range of different restriction enzymes to make the plasmid compatible with almost all of the most commonly used restriction enzymes, including those used in RFC10 compatible biobricks. <br />
<br><br><br />
A Gal1 inducible promoter is upstream of where the protein coding gene would be inserted. This promoter is strongly repressed by glucose and further allows the protein coding gene to be transcriptionally up-regulated upon the addition of galactose. Changing which of these two carbohydrates are present in the growth media therefore gives an enormous degree of control over the level of gene expression. Finally, a CYC1 terminator is present to ensure proper termination of transcription. <br />
<br />
<h3> BBa_K1322001- All-RFC Compatible Fluorescent Oscillator</h3><br />
<img src="https://static.igem.org/mediawiki/parts/9/9c/Sabinene_synthesis_pathway.gif" align="left" width="500" style="padding-bottom:0.5em; float:left" /><br />
<p><br />
As part of our collaboration with Vanderbilt Microfluidics, we were working with the existing biobrick K546546, which encodes a self-regulating fluorescent oscillating system. We wanted to make this part compatible with all major RFC standards, and did so by using our site-directed mutagenesis kit with specially designed primers. The sequence changes were designed so that the part should function equally well as it did before. For a gel showing confirmation, please see our lab notebook under October 11th. <br />
</p><br />
<br />
<h3> Sabinene Synthase </h3><br />
<br />
Although it is not yet RFC10 compatible and thus will not appear in the registry until later, we have successfully extracted the gene for sabinene synthase out of raw plant RNA. Following successful mutagenesis, we plan to add the biobrick prefix and suffixes so that this part can be made available at the registry for other iGEM teams to use.<br />
<br />
<br><br><br><br><br><br />
<h3>References:</h3><br />
<p><i>Scalcinati et al.: Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae. Microbial Cell Factories 2012 11:117<br><br />
Banerjee, Ecavade and Rao: Modulatory influence of sandalwood oil on mouse hepatic glutathione S-transferase activity and acid soluble sulphydryl level. Cancer Letters, 68 (1993) 105 - 109 <br />
Koch et al: Inhibitory effect of essential oils against herpes simplex virus type 2. Phytomedicine 2008;15(1-2):71-8. <br><br />
Rebecca Tirabassi, How to identify supercoils, nicks and circles in plasmid preps. Bitesizebio. October 8, 2014.</i><p></div>Hwangashttp://2014.igem.org/Team:Vanderbilt/PartsTeam:Vanderbilt/Parts2015-02-08T21:12:36Z<p>Hwangas: </p>
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<h3> BBa_K1322231- Optimized Santalene Synthase</h3><br />
<img src="https://static.igem.org/mediawiki/parts/f/f4/VU_Santalene_biosynthesis_path.gif" align="right" width="500" style="padding-bottom:0.5em; float:right" /><br />
<br />
BBa_K1322231 is a codon-optimized biobrick part encoding the gene for alpha-santalene synthase (EC 4.2.3.82). The enzyme catalyze the conversion of the common isoprenoid intermediate farnesyl pyrophosphate (FPP) into the sesquiterpene (+)-alpha-santelene in a single step. Traces of (-)-beta-santalene and bergamontene have previously been shown to be produced by this enzyme as well.<br />
<br><br><img src="https://static.igem.org/mediawiki/parts/0/0b/VU_San_psb_confirmation.jpg" align="left" width="340" style="padding-bottom:0.5em; float:left" /><br />
The gene, derived from a relative of the exotic sandalwood tree, has been demonstrated to produce functional terpene product in both yeast (Scalcinati et al 2012) and E. coli (data pending). This is possible due to several endogenous pathways that produce FPP as an intermediate, including the MEV and MEP pathways,<br />
<br><br><br />
In addition to being a prized fragrance, with what is often described as a warm, sweet woody scent, the sandalwood oil has been investigated for a number of other practical applications, including as a chemoprotective against carcinogenesis (Banaerjee, Ecavade, and Rao 1993) and inhibitors of viral reproduction (Koch et al 2008).<br />
<br><br><br />
Our biobrick has additional functionality added to it beyond just the coding sequence for santalene synthase. Immediately before the start codon is a yeast consensus sequence to permit efficient translation of the gene transcript in <i> S. cerevisiae</i>. Toward the end of the sequence there is also a sequence added inside the reading frame that encodes for a strep tag. The strep tag is a small, eight amino acid epitope tag that is translated onto the C terminus of the recombinant polypeptide. Its small size ensures that it will not likely interfere with protein function, yet in most situations it is still prominent enough that the common molecule streptavidin (in the form of Strep-tactin) can recognize and bind to it. Because anti-streptavidin antibodies are widely available, this opens the way for a range of possibilities, including simple confirmation assays of synthase expression by western blotting and quick purification of the synthase enzyme.<br />
<br><br><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/5/57/VU_pVU14006.png" align="right" alt="pVU14006" width="600" style="padding-bottom:0.5em; float:right" /><br />
We confirmed our part succesfully ligated into pSB1C3 by a diagnostic digest of miniprepped plasmid. While none of the samples had the santalene insert cleanly cut out, two observations were made which confirmed that in the fourth miniprepped sample, santalene had been successfully integrated. First, the size of the uncut fourth plasmid is equal to pSB1C3 plus the santalene synthase gene. Second, it was later noticed that the plasmids were grown in a non-demethylated strain of E. coli. This would explain why no inserts were cut out, since one enzyme used, ApaI, was dam methylation sensitive. This would also explain why the digested product is consistently larger than the undigested, since the linearized DNA should be above the uncut supercoiled form (Tirabassi 2014). <br />
<br />
<h3> pVU14006 </h3><br />
As a shuttle vector, pVU14006 is capable of expression both in E. coli and S. cerevisiae. It has resistance markers to both ampicillin and kanomycin, making selection convenient in both bacteria and yeast. For cloning in bacteria, it has a prokaryotic origin of replication taken out of pUC19. Two regions of base pair homology with the S. cerevisiae genome allow it to efficiently integrate into the yeast genome. Genomic integration has a number of advantages, including the potential for increased product yield. There is a multiple cloning site with a range of different restriction enzymes to make the plasmid compatible with almost all of the most commonly used restriction enzymes, including those used in RFC10 compatible biobricks. <br />
<br><br><br />
A Gal1 inducible promoter is upstream of where the protein coding gene would be inserted. This promoter is strongly repressed by glucose and further allows the protein coding gene to be transcriptionally up-regulated upon the addition of galactose. Changing which of these two carbohydrates are present in the growth media therefore gives an enormous degree of control over the level of gene expression. Finally, a CYC1 terminator is present to ensure proper termination of transcription. <br />
<br />
<h3> BBa_K1322001- All-RFC Compatible Fluorescent Oscillator</h3><br />
<img src="https://static.igem.org/mediawiki/parts/9/9c/Sabinene_synthesis_pathway.gif" align="left" width="500" style="padding-bottom:0.5em; float:left" /><br />
As part of our collaboration with Vanderbilt Microfluidics, we were working with the existing biobrick K546546, which encodes a self-regulating fluorescent oscillating system. We wanted to make this part compatible with all major RFC standards, and did so by using our site-directed mutagenesis kit with specially designed primers. The sequence changes were designed so that the part should function equally well as it did before. For a gel showing confirmation, please see our lab notebook under October 11th. <br />
<br />
<h3> Sabinene Synthase </h3><br />
<br />
Although it is not yet RFC10 compatible and thus will not appear in the registry until later, we have successfully extracted the gene for sabinene synthase out of raw plant RNA. Following successful mutagenesis, we plan to add the biobrick prefix and suffixes so that this part can be made available at the registry for other iGEM teams to use.<br />
<br />
<br><br><br><br><br><br />
<h3>References:</h3><br />
<p><i>Scalcinati et al.: Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae. Microbial Cell Factories 2012 11:117<br><br />
Banerjee, Ecavade and Rao: Modulatory influence of sandalwood oil on mouse hepatic glutathione S-transferase activity and acid soluble sulphydryl level. Cancer Letters, 68 (1993) 105 - 109 <br />
Koch et al: Inhibitory effect of essential oils against herpes simplex virus type 2. Phytomedicine 2008;15(1-2):71-8. <br><br />
Rebecca Tirabassi, How to identify supercoils, nicks and circles in plasmid preps. Bitesizebio. October 8, 2014.</i><p></div>Hwangashttp://2014.igem.org/Team:Vanderbilt/PartsTeam:Vanderbilt/Parts2015-02-08T21:12:12Z<p>Hwangas: </p>
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<h3> BBa_K1322231- Optimized Santalene Synthase</h3><br />
<img src="https://static.igem.org/mediawiki/parts/f/f4/VU_Santalene_biosynthesis_path.gif" align="right" width="500" style="padding-bottom:0.5em; float:right" /><br />
<br />
BBa_K1322231 is a codon-optimized biobrick part encoding the gene for alpha-santalene synthase (EC 4.2.3.82). The enzyme catalyze the conversion of the common isoprenoid intermediate farnesyl pyrophosphate (FPP) into the sesquiterpene (+)-alpha-santelene in a single step. Traces of (-)-beta-santalene and bergamontene have previously been shown to be produced by this enzyme as well.<br />
<br><br><img src="https://static.igem.org/mediawiki/parts/0/0b/VU_San_psb_confirmation.jpg" align="left" width="340" style="padding-bottom:0.5em; float:left" /><br />
The gene, derived from a relative of the exotic sandalwood tree, has been demonstrated to produce functional terpene product in both yeast (Scalcinati et al 2012) and E. coli (data pending). This is possible due to several endogenous pathways that produce FPP as an intermediate, including the MEV and MEP pathways,<br />
<br><br><br />
In addition to being a prized fragrance, with what is often described as a warm, sweet woody scent, the sandalwood oil has been investigated for a number of other practical applications, including as a chemoprotective against carcinogenesis (Banaerjee, Ecavade, and Rao 1993) and inhibitors of viral reproduction (Koch et al 2008).<br />
<br><br><br />
Our biobrick has additional functionality added to it beyond just the coding sequence for santalene synthase. Immediately before the start codon is a yeast consensus sequence to permit efficient translation of the gene transcript in <i> S. cerevisiae</i>. Toward the end of the sequence there is also a sequence added inside the reading frame that encodes for a strep tag. The strep tag is a small, eight amino acid epitope tag that is translated onto the C terminus of the recombinant polypeptide. Its small size ensures that it will not likely interfere with protein function, yet in most situations it is still prominent enough that the common molecule streptavidin (in the form of Strep-tactin) can recognize and bind to it. Because anti-streptavidin antibodies are widely available, this opens the way for a range of possibilities, including simple confirmation assays of synthase expression by western blotting and quick purification of the synthase enzyme.<br />
<br><br><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/5/57/VU_pVU14006.png" align="right" alt="pVU14006" width="600" style="padding-bottom:0.5em; float:right" /><br />
We confirmed our part succesfully ligated into pSB1C3 by a diagnostic digest of miniprepped plasmid. While none of the samples had the santalene insert cleanly cut out, two observations were made which confirmed that in the fourth miniprepped sample, santalene had been successfully integrated. First, the size of the uncut fourth plasmid is equal to pSB1C3 plus the santalene synthase gene. Second, it was later noticed that the plasmids were grown in a non-demethylated strain of E. coli. This would explain why no inserts were cut out, since one enzyme used, ApaI, was dam methylation sensitive. This would also explain why the digested product is consistently larger than the undigested, since the linearized DNA should be above the uncut supercoiled form (Tirabassi 2014). <br />
<br />
<h3> pVU14006 </h3><br />
As a shuttle vector, pVU14006 is capable of expression both in E. coli and S. cerevisiae. It has resistance markers to both ampicillin and kanomycin, making selection convenient in both bacteria and yeast. For cloning in bacteria, it has a prokaryotic origin of replication taken out of pUC19. Two regions of base pair homology with the S. cerevisiae genome allow it to efficiently integrate into the yeast genome. Genomic integration has a number of advantages, including the potential for increased product yield. There is a multiple cloning site with a range of different restriction enzymes to make the plasmid compatible with almost all of the most commonly used restriction enzymes, including those used in RFC10 compatible biobricks. <br />
<br><br><br />
A Gal1 inducible promoter is upstream of where the protein coding gene would be inserted. This promoter is strongly repressed by glucose and further allows the protein coding gene to be transcriptionally up-regulated upon the addition of galactose. Changing which of these two carbohydrates are present in the growth media therefore gives an enormous degree of control over the level of gene expression. Finally, a CYC1 terminator is present to ensure proper termination of transcription. <br />
<br />
<h3> BBa_K1322001- All-RFC Compatible Fluorescent Oscillator</h3><br />
<br />
As part of our collaboration with Vanderbilt Microfluidics, we were working with the existing biobrick K546546, which encodes a self-regulating fluorescent oscillating system. We wanted to make this part compatible with all major RFC standards, and did so by using our site-directed mutagenesis kit with specially designed primers. The sequence changes were designed so that the part should function equally well as it did before. For a gel showing confirmation, please see our lab notebook under October 11th. <br />
<img src="https://static.igem.org/mediawiki/parts/9/9c/Sabinene_synthesis_pathway.gif" align="left" width="500" style="padding-bottom:0.5em; float:left" /><br />
<h3> Sabinene Synthase </h3><br />
<br />
Although it is not yet RFC10 compatible and thus will not appear in the registry until later, we have successfully extracted the gene for sabinene synthase out of raw plant RNA. Following successful mutagenesis, we plan to add the biobrick prefix and suffixes so that this part can be made available at the registry for other iGEM teams to use.<br />
<br />
<br><br><br><br><br><br />
<h3>References:</h3><br />
<p><i>Scalcinati et al.: Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae. Microbial Cell Factories 2012 11:117<br><br />
Banerjee, Ecavade and Rao: Modulatory influence of sandalwood oil on mouse hepatic glutathione S-transferase activity and acid soluble sulphydryl level. Cancer Letters, 68 (1993) 105 - 109 <br />
Koch et al: Inhibitory effect of essential oils against herpes simplex virus type 2. Phytomedicine 2008;15(1-2):71-8. <br><br />
Rebecca Tirabassi, How to identify supercoils, nicks and circles in plasmid preps. Bitesizebio. October 8, 2014.</i><p></div>Hwangashttp://2014.igem.org/Team:Vanderbilt/PartsTeam:Vanderbilt/Parts2015-02-08T21:11:47Z<p>Hwangas: </p>
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<h3> BBa_K1322231- Optimized Santalene Synthase</h3><br />
<img src="https://static.igem.org/mediawiki/parts/f/f4/VU_Santalene_biosynthesis_path.gif" align="right" width="500" style="padding-bottom:0.5em; float:right" /><br />
<br />
BBa_K1322231 is a codon-optimized biobrick part encoding the gene for alpha-santalene synthase (EC 4.2.3.82). The enzyme catalyze the conversion of the common isoprenoid intermediate farnesyl pyrophosphate (FPP) into the sesquiterpene (+)-alpha-santelene in a single step. Traces of (-)-beta-santalene and bergamontene have previously been shown to be produced by this enzyme as well.<br />
<br><br><img src="https://static.igem.org/mediawiki/parts/0/0b/VU_San_psb_confirmation.jpg" align="left" width="340" style="padding-bottom:0.5em; float:left" /><br />
The gene, derived from a relative of the exotic sandalwood tree, has been demonstrated to produce functional terpene product in both yeast (Scalcinati et al 2012) and E. coli (data pending). This is possible due to several endogenous pathways that produce FPP as an intermediate, including the MEV and MEP pathways,<br />
<br><br><br />
In addition to being a prized fragrance, with what is often described as a warm, sweet woody scent, the sandalwood oil has been investigated for a number of other practical applications, including as a chemoprotective against carcinogenesis (Banaerjee, Ecavade, and Rao 1993) and inhibitors of viral reproduction (Koch et al 2008).<br />
<br><br><br />
Our biobrick has additional functionality added to it beyond just the coding sequence for santalene synthase. Immediately before the start codon is a yeast consensus sequence to permit efficient translation of the gene transcript in <i> S. cerevisiae</i>. Toward the end of the sequence there is also a sequence added inside the reading frame that encodes for a strep tag. The strep tag is a small, eight amino acid epitope tag that is translated onto the C terminus of the recombinant polypeptide. Its small size ensures that it will not likely interfere with protein function, yet in most situations it is still prominent enough that the common molecule streptavidin (in the form of Strep-tactin) can recognize and bind to it. Because anti-streptavidin antibodies are widely available, this opens the way for a range of possibilities, including simple confirmation assays of synthase expression by western blotting and quick purification of the synthase enzyme.<br />
<br><br><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/5/57/VU_pVU14006.png" align="right" alt="pVU14006" width="600" style="padding-bottom:0.5em; float:right" /><br />
We confirmed our part succesfully ligated into pSB1C3 by a diagnostic digest of miniprepped plasmid. While none of the samples had the santalene insert cleanly cut out, two observations were made which confirmed that in the fourth miniprepped sample, santalene had been successfully integrated. First, the size of the uncut fourth plasmid is equal to pSB1C3 plus the santalene synthase gene. Second, it was later noticed that the plasmids were grown in a non-demethylated strain of E. coli. This would explain why no inserts were cut out, since one enzyme used, ApaI, was dam methylation sensitive. This would also explain why the digested product is consistently larger than the undigested, since the linearized DNA should be above the uncut supercoiled form (Tirabassi 2014). <br />
<br />
<h3> pVU14006 </h3><br />
As a shuttle vector, pVU14006 is capable of expression both in E. coli and S. cerevisiae. It has resistance markers to both ampicillin and kanomycin, making selection convenient in both bacteria and yeast. For cloning in bacteria, it has a prokaryotic origin of replication taken out of pUC19. Two regions of base pair homology with the S. cerevisiae genome allow it to efficiently integrate into the yeast genome. Genomic integration has a number of advantages, including the potential for increased product yield. There is a multiple cloning site with a range of different restriction enzymes to make the plasmid compatible with almost all of the most commonly used restriction enzymes, including those used in RFC10 compatible biobricks. <br />
<br><br><br />
A Gal1 inducible promoter is upstream of where the protein coding gene would be inserted. This promoter is strongly repressed by glucose and further allows the protein coding gene to be transcriptionally up-regulated upon the addition of galactose. Changing which of these two carbohydrates are present in the growth media therefore gives an enormous degree of control over the level of gene expression. Finally, a CYC1 terminator is present to ensure proper termination of transcription. <br />
<br />
<h3> BBa_K1322001- All-RFC Compatible Fluorescent Oscillator</h3><br />
<br />
As part of our collaboration with Vanderbilt Microfluidics, we were working with the existing biobrick K546546, which encodes a self-regulating fluorescent oscillating system. We wanted to make this part compatible with all major RFC standards, and did so by using our site-directed mutagenesis kit with specially designed primers. The sequence changes were designed so that the part should function equally well as it did before. For a gel showing confirmation, please see our lab notebook under October 11th. <br />
<img src="https://static.igem.org/mediawiki/parts/9/9c/Sabinene_synthesis_pathway.gif" align="left" width="500" style="padding-bottom:0.5em; float:left" /><br />
<h3> Sabinene Synthase </h3><br />
<br />
Although it is not yet RFC10 compatible and thus will not appear in the registry until later, we have successfully extracted the gene for sabinene synthase out of raw plant RNA. Following successful mutagenesis, we plan to add the biobrick prefix and suffixes so that this part can be made available at the registry for other iGEM teams to use.<br />
<br />
<br><br><br><br><br><br />
<h3>References:</h3><br />
<br><br />
<p>Scalcinati et al.: Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae. Microbial Cell Factories 2012 11:117<br><br />
Banerjee, Ecavade and Rao: Modulatory influence of sandalwood oil on mouse hepatic glutathione S-transferase activity and acid soluble sulphydryl level. Cancer Letters, 68 (1993) 105 - 109 <br />
Koch et al: Inhibitory effect of essential oils against herpes simplex virus type 2. Phytomedicine 2008;15(1-2):71-8. <br><br />
Rebecca Tirabassi, How to identify supercoils, nicks and circles in plasmid preps. Bitesizebio. October 8, 2014.<p></div>Hwangashttp://2014.igem.org/Team:Vanderbilt/ProjectTeam:Vanderbilt/Project2015-02-08T21:10:52Z<p>Hwangas: </p>
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<td><h3> Introduction </h3><br />
<p> <br />
<figure><br />
<img src="https://static.igem.org/mediawiki/parts/5/55/VU_greenhouse_plants.JPG" align="right" alt="Plants growing in the Vanderbilt greenhouse" height="300" width="300"/><br />
</figure><br />
<p><br />
The production of plant essential oils and their derivatives represents an over 9 billion dollar industry when considering just their applications in the food and fragrance industries <sup>1</sup>. A staggering 23 million kilograms of citrus oil alone are produced worldwide each year. Up until only a couple decades ago, the production of these essential oils was done exclusively by chemical extraction from plant material. However, the sudden emergence of synthetic biology a versatile and efficient tool has the potential to transform this immense industry, the products of which nearly everyone will come in contact with on a daily basis. <br />
</p><br />
<p><br />
By harnessing existing biosynthetic pathways and introducing enzymes taken from plants into more malleable model systems, it will be possible to significantly improve on current methods of the active components of essential oils, most notably the terpenoids. While most plants express terpenes in the range of parts per million and thus require very large scale operations to be commercially viable, early forays into the biological production of terpenes have proven that it is possible to improve yields 100-fold <sup>2</sup>. We selected a total of nine different terpenes to produce (see table), each of which has practical applications which make them prime candidates for alternate means of production. The first aspect of our project involves using the great potential of synthetic biology to design a commercially-viable strategy for the production of any class of terpenoid.<br />
</p><br />
<p><br />
Just as important to the economic benefit of this approach is its environment benefit. With chemical terpene extraction being such a relatively inefficient process, it is necessary to process large amounts of plant material to get a substantive yield. This may not pose as significant of an issue for citrus growers, but many of the most prized compounds are taken from the rarest species of plant. The continuation of the status quo in terms of terpene extraction is not an environmentally sustainable solution. In our selection of terpenes, we placed a large emphasis on choosing compounds from some of the most rare species possible.<br />
</p><br />
<p><br />
The best example of this idea behind our project can be seen in the gene for santalene synthase. The only species know to have genes to produce this terpene are found in remote regions of India and Australia, and one of them is listed as a vulnerable species by the IUCN. The trees can live for hundreds of years, but are the target of widespread over-exploitation, to the point that, for example, the Indian government has banned the export of sandalwood. Synthetic biology can produce the exact same active ingredients of sandalwood oil in a way that is both more economically and environmentally sound. <br />
</p><br />
<p><br />
In order for our idea to truly be applicable to exotic and endangered species of plant, we had to take an approach that was quite different from the one most iGEM teams have historically taken. What we were looking for was a quick, inexpensive method of cloning genes that was also compatible with species that have not had their entire genomes sequenced. Often, iGEM teams resort to synthesizing their genes through third parties. However, this can be fairly costly especially given the moderately large size of many of these synthase genes, may take several weeks or even a month to finish synthesizing, and cannot be done unless the gene's sequence is known in its entirety. By going back to basics and taking raw plants as our source material, we were able to avoid these issues and demonstrated how our approach was a practically viable one. <br />
</p><br />
<br />
<table width="500" border="1" cellpadding="5"><br />
<br />
<tr><br />
<td align="center" valign="center"><br />
<img src="https://static.igem.org/mediawiki/parts/f/f3/VU_Cadenine.png"width="200" /><br />
<br /><br />
<b>Cadenine</b><br><br><br />
Main essential oil: Cade oil<br><br />
Applications: Antifungal, bactericidal, and antioxidant<br><br />
Plant Species: <i>Gossypium hirsutum</i> (cotton)<br><br />
Synthase Gene: δ-cadenine synthase (E.C. 4.2.3.13)<br />
</td><br />
<td align="center" valign="center"><br />
<img src="https://static.igem.org/mediawiki/parts/6/67/VU_Carene.png" width="200" /><br />
<br /><br />
<b>Carene</b><br><br><br />
Main essential oils: Rosemary and Cedar oil<br><br />
Applications: Insecticide, anti-inflammatory, and central nervous system depressant<br><br />
Plant Species: <i>Picea abies</i> (norway spruce)<br><br />
Synthase Gene: carene synthase (E.C. 4.2.3.107)<br />
</td><br />
<td align="center" valign="center"><br />
<img src="https://static.igem.org/mediawiki/parts/1/13/VU_Humelene.png" width="200" /><br />
<br /><br />
<b>Humelene</b><br><br><br />
Main essential oils: Hops oil<br><br />
Applications: Culinary spice, and anti-inflammatory<br><br />
Plant Species: <i>Zingiber zerumbet</i> (shampoo ginger)<br><br />
Synthase Gene: α-humulene synthase (E.C. 4.2.3.104)<br />
</td><br />
</tr><br />
<tr><br />
<td align="center" valign="center"><br />
<img src="https://static.igem.org/mediawiki/parts/6/68/VU_myrcene.png" width="200" /><br />
<br /><br />
<b>Myrcene</b><br><br><br />
Main essential oils: Thyme and Hops oil <br><br />
Applications: Fragrance, analgesic, and anti-inflammatory<br><br />
Plant Species: <i>Perilla frutescens</i> (Green Shiso)<br><br />
Synthase Gene: myrcene synthase (E.C. 4.2.3.15)<br />
</td><br />
<td align="center" valign="center"><br />
<img src="https://static.igem.org/mediawiki/parts/7/7c/VU_R_linalool.png" width="200" /><br />
<br /><br />
<b>(R)-Linalool</b><br><br><br />
Main essential oils: Lavender oil<br><br />
Applications: Fragrance, and insecticide<br><br />
Plant Species: <i>Mentha citrata</i> (lemon mint)<br><br />
Synthase Gene: (R)-linalool synthase (E.C. 4.2.3.26)<br />
</td><br />
<td align="center" valign="center"><br />
<img src="https://static.igem.org/mediawiki/parts/7/7a/VU_S_linalool.png" width="200" /><br />
<br /><br />
<b>(S)-Linalool</b><br><br><br />
Main essential oils: Citrus and Coriander oil<br><br />
Applications: Fragrance, and insecticide<br><br />
Plant Species Chosen: <i>Arabidopsis thaliana</i> (thale cress)<br><br />
Synthase Gene: (S)-linalool synthase (E.C. 4.2.3.25)<br />
</td><br />
</tr><br />
<tr><br />
<td align="center" valign="center"><br />
<img src="https://static.igem.org/mediawiki/parts/e/e8/VU_Sabinene.png" width="200" /><br />
<br /><br />
<b>Sabinene</b><br><br><br />
Main essential oils: Junier Coriander oil<br><br />
Applications: Spice, and antimicrobial<br><br />
Plant Species Chosen: <i> Salvia officinalis</i> (sage)<br><br />
Synthase Gene: (+)-sabinene synthase (E.C. 4.2.3.110) <br />
</td><br />
<td align="center" valign="center"><br />
<img src="https://static.igem.org/mediawiki/parts/5/54/VU_Santalene.png" width="200" /><br />
<br /><br />
<b>Santalene</b><br><br><br />
Main essential oils: Sandalwood oil<br><br />
Applications: Fragrance, antiviral, and tumor-suppressant<br><br />
Plant Species Chosen: <i> Santalum album</i> (sandalwood tree)<br><br />
Synthase Gene: α-santalene synthase (E.C. 4.2.3.82)<br />
</td><br />
<td align="center" valign="center"><br />
<img src="https://static.igem.org/mediawiki/parts/8/8d/VU_Zingiberene.png" width="200" /><br />
<br /><br />
<b>Zingiberene</b><br><br><br />
Main essential oils: Ginger oil<br><br />
Applications: Flavoring and pesticide<br><br />
Plant Species Chosen: <i> Ocimum basilicum</i> (basil)<br><br />
Synthase Gene: α-zingiberene synthase (E.C. 4.2.3.65)<br />
</td><br />
</tr><br />
</table><br />
<br />
<tr> <br />
<td><br />
<h3>Design</h3><br />
<img src="https://static.igem.org/mediawiki/parts/e/ef/Terpenoid_biosynthesis_pathway.png" align=right alt="terepnoid biosynthesis pathways" width="300" height="300" style="padding-bottom:0.5em; float:right" /><br />
<p><br />
Terpene biosynthesis in plants is part of larger pathways that metabolize isoprenoid intermediates. Genes encoding for enzymes known as synthases catalyze the terminal step in these pathways, from a precursor (commonly farnesyl pyrophosphate (FPP) or garnyl pyrophosphate (GPP)) to the final terpene product. As it happens, two well established and genetically manipulable model organisms- the bacterium <i> Escherichia coli </i> and baker's yeast <i> Saccharomyces cerevisiae</i>- produce moderate amounts of GPP and FPP as part of their endogenous non-mevalonate pathway (MEP) and mevalonate pathway (MEV) respectively<sup>3</sup>. All that is required for either of these organisms to begin producing terepenes is to introduce that single synthase gene.<br />
</p><br />
<p><br />
Yeast initially was our main target for a few advantages it appeared to have as a production platform. First, the MEV pathway is found in all eukaryotes including plants and fungi, so better yield were expected by choosing it over the MEP pathway in prokaryotes<sup>4</sup>. Second, it would be possible to physically integrate our gene into one of the yeast's chromosomes by using homologous recombination. An inducible promoter could be included to further increase production. Third, as a diploid the yeast could be made homozygous for the terpene gene. We soon found out that no exiting vector had all of the features we would want. Therefore, we designed our own new plasmid vector, pVU140006, that contained a number of important features and advances over previous plasmids for this purpose. See our parts page for more information on what special features we included and their relevance to the project<br />
</p><br />
<br />
</tr><br />
<tr><br />
<td><h3>Methods</h3><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/8/82/VU_experiment_1_diagram.png" align=left alt="First few experiments" width="300" style="padding-bottom:0.5em; float:right" /><br />
<p><br />
Our project had several co-dependent sub-project that were all worked on in parallel. These can roughly be divided into two categories: the first involving work on our synthase genes and the second involving the construction of a new, specially designed plasmid vector. We tried two different team structures over the year to see which would give the best results. For the Spring, we had the original idea of dividing members into independent groups, each working on a specific terpene. Each group was headed by a single group manager who would teach 4-5 new members the protocol that was to be preformed and then supervise that the work was carried out correctly. On occasion either the group managers or the organization president or wetware director would also given lessons to teach members about the techniques and theory involved at each step. All group managers were in turn trained by either the president or wetware director, both of whom had come with the experiment, acquired all the necessary primers and reagents, wrote up the protocol, and had preformed it prior to any group-phase work for the purposes of troubleshooting and predicting where issues may come up. The president or wetware director also helped the group manager in being present during all experiments for answering questions, preparing materials, and other forms of assistance. <br />
</p><br />
<p><br />
Each group first planted seeds under the appropriate soil, humidity, and temperature conditions at the Vanderbilt Greenhouse. Once the majority of these grew into saplings with green leaves, Samples were flash frozen in liquid nitrogen in preparation for a genomic DNA extraction. After the extractions, nanodrop concentration readings and agarose gels confirmed the presence of high molecular weight genomic DNA. Groups then ran a PCR on their genomic DNA with primers specific to their synthase gene. More gels were run to check for PCR product. At this point, the semester was coming to an end, so groups were disbanded before most managed to isolate their synthase gene. Over the summer, the president and wetware director continued troubleshooting those genes which were not amplifying and eventually got each to the point where consistent PCR product bands were produced. </p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/f/fd/VU_experiment_2_diagram.png" align=right alt="First few experiments" width="300" style="padding-bottom:0.5em; float:right" /><br />
<br />
<p><br />
Once we had clear banding patterns, it became clear that the number of introns in each of our genes (a variable which was unknown since most of the plants we worked with have not had their genomes sequenced) was too great for cloning and expression to be practical. Therefore, as soon as the fall semester began, we shifted strategy to isolating RNA from our plants. This RNA could be converted to cDNA by reverse transcription, which would eliminate the issue with introns we were having. Several of our greenhouse plants were no longer available, so we reduced the number of target terpenes we were focusing on. After extracting RNA and running an RT-PCR, several samples produced bands that corresponded roughly to where the synthase gene should be. These were gel extracted. Because almost every synthase gene had restriction sites in them that prevented them from being RFC10 compatible, we ligated the genes in pUC19 for site directed mutagenesis. After that, a second processing step would have been necessary to add the correct restriction sites to each gene to allow them to be integrated into pSB1C3 as a biobrick. In the interest of time, we synthesized a codon-optimized santalene synthase gene in order to skip these RFC10 processing steps, even though we had already successfully reverse transcribed cDNA of the santalene synthase gene.<br />
</p><br />
<p><br />
In the spring concurrent to work by the terpene groups, work began on plasmid construction. This was preformed by the president, wetware director, and a handful of others rather than in group format. Each gene cassette for our final plasmid was first identified in an existing, readily available plasmid. All of these cassettes were extracted by PCR using those plasmids as templates. Overlap extension PCR was then done on the gel-purified product to add restriction sites and homology regions for the purposes of eventually combining all of the cassettes together into a single plasmid. By the end of the summer, only one final fragment remained to be inserted to complete the intermediate plasmid pVU14004. Upon the successful creation of pVU14004, several restriction enzyme sites had to be removed by site directed mutagensis in order to make the plasmid RFC10 compatible. <br />
</p><br />
<br />
<h3> Results and Directions </h3><br />
<p><br />
Several factors contributed to the difficulty we experienced during the final phase of the project. First, member engagement suffered a significant decline between the spring and fall semesters, to the point where only a small handful of people were left to preform all experiments. Second, the late realization that we had to change our cloning strategy to modified cDNA inserts effectively meant we had to start anew in late August despite having what was a good head start when we began in early March. Third, the RFC10 requirements added a substantial dimension of difficulty to the project since all of our starting material (both the extracted gene cassettes for plasmid construction and the synthase genes) contained multiple sites that made them incompatible with the biobrick standard. Nevertheless, our team accomplished an enormous amount during our first year in competition. </p><br />
<br><br />
<table width="90%"><br />
<tr><br />
<td> <i>Terpene </i> </td><br />
<td> <i>Plant Genomic DNA Successfully Extracted</i> </td><br />
<td> <i>Synthase Gene Successfully PCR Isolated </i> </td><br />
<td> <i>Plant RNA Successfully Extracted</i> </td><br />
<td> <i>Synthase gene Successfully Reverse Transcribed</i> </td><br />
<td> <i>Terpene Successfully Produced in E. coli or yeast <i> </td><br />
<tr><br />
<td> <b>Cadinene</b> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td> <br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td> <br />
</tr><br />
<tr><br />
<td> <b>Carene</b> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td> <br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td> <br />
</tr><br />
<tr><br />
<td> <b>Humelene</b> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td> <br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td> <br />
</tr><br />
<tr><br />
<td> <b>Myrcene</b> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td> <br />
</tr><br />
<tr><br />
<td> <b>(R)-Linalool</b> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td> <br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td> <br />
</tr><br />
<tr><br />
<td> <b>(S)-Linalool</b> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td> <br />
</tr><br />
<tr><br />
<td> <b>Sabinene</b> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5b/Yellow_square.png" width="50"> </td> <br />
</tr><br />
<tr><br />
<td> <b>Santalene</b> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5b/Yellow_square.png" width="50"> </td><br />
</tr><br />
<tr><br />
<td> <b>Zingiberene</b> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td> <br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td> <br />
</tr><br />
</table><br />
<br />
<br><br />
<img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="100" align="right"><br />
<p><br />
We successfully constructed pVU14004, which contains all of the cassettes needed to have all of functionality we had intended. However, this form of the plasmid has XbaI and EcoRI sites that make it incompatible with RFC10. These sites were successfully changed to missense mutations by site directed mutagenesis. The final step required to make pVU14006 is changing the multiple cloning site (MCS) to have the biobrick prefix and suffix. In its current form, the plasmid has a functional MCS but it does not yet have the specific order of EcoRI, XbaI, SpeI, and PstI sites required by RFC10. <br />
</p><br />
<p><br />
For a listing of all the medal requirements we successfully fulfilled over the course of our project, please visit <a href="https://2014.igem.org/Team:Vanderbilt/Project/Requirements"style="color:#000000"> <u> this page </u></a>.<br />
</p><br />
<br />
<h3> Collaborations </h3><br />
<img src="https://static.igem.org/mediawiki/parts/6/64/Ravenwood_high_school.jpg" width="300" align="left"><br />
<p><br />
In addition to our own wetware project, our team led fruitful collaborations with a total of three other iGEM teams. First, we played a major role in assisting Vanderbilt's microfluidic division with the biological aspect of their project. We prepared the biobrick parts they tested in their microfluidic device, including transforming the E. coli they used to study their quorum-sensing fluorescent oscillator circuit. Second, we provided feedback to Vanderbilt's software division about their own project involving a program to aid in the manipulation of genetic sequences. We used the program as if it were for a real project and gave them suggestions on how to make their program easier to use and more useful to biologists.<br />
</p><br />
<br />
<p> <br />
Last but not least, we provided significant assistance and guidance to the Ravenwood Raptors high school iGEM team. In a series of conversations with Dr. Amanda Benson, we planned a smaller scale version of our own project that involved only a single terpene synthase gene. After visiting the high school and presenting our idea, the students voted to choose it for their project. We also provided the primers and sage genomic DNA template they used in their experiments. <br />
</p><br />
<br />
<br><br />
<b> References: </b></br><br />
<i>1. USDA Industrial Uses Reports. Essential Oils Widely Used in Flavors and Fragrances. September 1995. <br><br />
2. Ajikumar PK, Tyo K, Carlsen S, Mucha O, Phon TH, Stephanopoulos G. Terpenoids: opportunities for biosynthesis of natural product drugs using engineered microorganisms. Mol Pharm. 2008;5(2):167-90. <br><br />
3. Dudareva N, Klempien A, Muhlemann JK, Kaplan I. Biosynthesis, function and metabolic engineering of plant volatile organic compounds. New Phytol. 2013;198(1):16-32. <br><br />
4.Martin VJ, Pitera DJ, Withers ST, Newman JD, Keasling JD. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol. 2003;21(7):796-802. <br><br />
</i><br />
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<td><h3> Introduction </h3><br />
<p> <br />
<figure><br />
<img src="https://static.igem.org/mediawiki/parts/5/55/VU_greenhouse_plants.JPG" align="right" alt="Plants growing in the Vanderbilt greenhouse" height="420" width="300"/><br />
</figure><br />
<p><br />
The production of plant essential oils and their derivatives represents an over 9 billion dollar industry when considering just their applications in the food and fragrance industries <sup>1</sup>. A staggering 23 million kilograms of citrus oil alone are produced worldwide each year. Up until only a couple decades ago, the production of these essential oils was done exclusively by chemical extraction from plant material. However, the sudden emergence of synthetic biology a versatile and efficient tool has the potential to transform this immense industry, the products of which nearly everyone will come in contact with on a daily basis. <br />
</p><br />
<p><br />
By harnessing existing biosynthetic pathways and introducing enzymes taken from plants into more malleable model systems, it will be possible to significantly improve on current methods of the active components of essential oils, most notably the terpenoids. While most plants express terpenes in the range of parts per million and thus require very large scale operations to be commercially viable, early forays into the biological production of terpenes have proven that it is possible to improve yields 100-fold <sup>2</sup>. We selected a total of nine different terpenes to produce (see table), each of which has practical applications which make them prime candidates for alternate means of production. The first aspect of our project involves using the great potential of synthetic biology to design a commercially-viable strategy for the production of any class of terpenoid.<br />
</p><br />
<p><br />
Just as important to the economic benefit of this approach is its environment benefit. With chemical terpene extraction being such a relatively inefficient process, it is necessary to process large amounts of plant material to get a substantive yield. This may not pose as significant of an issue for citrus growers, but many of the most prized compounds are taken from the rarest species of plant. The continuation of the status quo in terms of terpene extraction is not an environmentally sustainable solution. In our selection of terpenes, we placed a large emphasis on choosing compounds from some of the most rare species possible.<br />
</p><br />
<p><br />
The best example of this idea behind our project can be seen in the gene for santalene synthase. The only species know to have genes to produce this terpene are found in remote regions of India and Australia, and one of them is listed as a vulnerable species by the IUCN. The trees can live for hundreds of years, but are the target of widespread over-exploitation, to the point that, for example, the Indian government has banned the export of sandalwood. Synthetic biology can produce the exact same active ingredients of sandalwood oil in a way that is both more economically and environmentally sound. <br />
</p><br />
<p><br />
In order for our idea to truly be applicable to exotic and endangered species of plant, we had to take an approach that was quite different from the one most iGEM teams have historically taken. What we were looking for was a quick, inexpensive method of cloning genes that was also compatible with species that have not had their entire genomes sequenced. Often, iGEM teams resort to synthesizing their genes through third parties. However, this can be fairly costly especially given the moderately large size of many of these synthase genes, may take several weeks or even a month to finish synthesizing, and cannot be done unless the gene's sequence is known in its entirety. By going back to basics and taking raw plants as our source material, we were able to avoid these issues and demonstrated how our approach was a practically viable one. <br />
</p><br />
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<table width="500" border="1" cellpadding="5"><br />
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<td align="center" valign="center"><br />
<img src="https://static.igem.org/mediawiki/parts/f/f3/VU_Cadenine.png"width="200" /><br />
<br /><br />
<b>Cadenine</b><br><br><br />
Main essential oil: Cade oil<br><br />
Applications: Antifungal, bactericidal, and antioxidant<br><br />
Plant Species: <i>Gossypium hirsutum</i> (cotton)<br><br />
Synthase Gene: δ-cadenine synthase (E.C. 4.2.3.13)<br />
</td><br />
<td align="center" valign="center"><br />
<img src="https://static.igem.org/mediawiki/parts/6/67/VU_Carene.png" width="200" /><br />
<br /><br />
<b>Carene</b><br><br><br />
Main essential oils: Rosemary and Cedar oil<br><br />
Applications: Insecticide, anti-inflammatory, and central nervous system depressant<br><br />
Plant Species: <i>Picea abies</i> (norway spruce)<br><br />
Synthase Gene: carene synthase (E.C. 4.2.3.107)<br />
</td><br />
<td align="center" valign="center"><br />
<img src="https://static.igem.org/mediawiki/parts/1/13/VU_Humelene.png" width="200" /><br />
<br /><br />
<b>Humelene</b><br><br><br />
Main essential oils: Hops oil<br><br />
Applications: Culinary spice, and anti-inflammatory<br><br />
Plant Species: <i>Zingiber zerumbet</i> (shampoo ginger)<br><br />
Synthase Gene: α-humulene synthase (E.C. 4.2.3.104)<br />
</td><br />
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<tr><br />
<td align="center" valign="center"><br />
<img src="https://static.igem.org/mediawiki/parts/6/68/VU_myrcene.png" width="200" /><br />
<br /><br />
<b>Myrcene</b><br><br><br />
Main essential oils: Thyme and Hops oil <br><br />
Applications: Fragrance, analgesic, and anti-inflammatory<br><br />
Plant Species: <i>Perilla frutescens</i> (Green Shiso)<br><br />
Synthase Gene: myrcene synthase (E.C. 4.2.3.15)<br />
</td><br />
<td align="center" valign="center"><br />
<img src="https://static.igem.org/mediawiki/parts/7/7c/VU_R_linalool.png" width="200" /><br />
<br /><br />
<b>(R)-Linalool</b><br><br><br />
Main essential oils: Lavender oil<br><br />
Applications: Fragrance, and insecticide<br><br />
Plant Species: <i>Mentha citrata</i> (lemon mint)<br><br />
Synthase Gene: (R)-linalool synthase (E.C. 4.2.3.26)<br />
</td><br />
<td align="center" valign="center"><br />
<img src="https://static.igem.org/mediawiki/parts/7/7a/VU_S_linalool.png" width="200" /><br />
<br /><br />
<b>(S)-Linalool</b><br><br><br />
Main essential oils: Citrus and Coriander oil<br><br />
Applications: Fragrance, and insecticide<br><br />
Plant Species Chosen: <i>Arabidopsis thaliana</i> (thale cress)<br><br />
Synthase Gene: (S)-linalool synthase (E.C. 4.2.3.25)<br />
</td><br />
</tr><br />
<tr><br />
<td align="center" valign="center"><br />
<img src="https://static.igem.org/mediawiki/parts/e/e8/VU_Sabinene.png" width="200" /><br />
<br /><br />
<b>Sabinene</b><br><br><br />
Main essential oils: Junier Coriander oil<br><br />
Applications: Spice, and antimicrobial<br><br />
Plant Species Chosen: <i> Salvia officinalis</i> (sage)<br><br />
Synthase Gene: (+)-sabinene synthase (E.C. 4.2.3.110) <br />
</td><br />
<td align="center" valign="center"><br />
<img src="https://static.igem.org/mediawiki/parts/5/54/VU_Santalene.png" width="200" /><br />
<br /><br />
<b>Santalene</b><br><br><br />
Main essential oils: Sandalwood oil<br><br />
Applications: Fragrance, antiviral, and tumor-suppressant<br><br />
Plant Species Chosen: <i> Santalum album</i> (sandalwood tree)<br><br />
Synthase Gene: α-santalene synthase (E.C. 4.2.3.82)<br />
</td><br />
<td align="center" valign="center"><br />
<img src="https://static.igem.org/mediawiki/parts/8/8d/VU_Zingiberene.png" width="200" /><br />
<br /><br />
<b>Zingiberene</b><br><br><br />
Main essential oils: Ginger oil<br><br />
Applications: Flavoring and pesticide<br><br />
Plant Species Chosen: <i> Ocimum basilicum</i> (basil)<br><br />
Synthase Gene: α-zingiberene synthase (E.C. 4.2.3.65)<br />
</td><br />
</tr><br />
</table><br />
<br />
<tr> <br />
<td><br />
<h3>Design</h3><br />
<img src="https://static.igem.org/mediawiki/parts/e/ef/Terpenoid_biosynthesis_pathway.png" align=right alt="terepnoid biosynthesis pathways" width="300" height="300" style="padding-bottom:0.5em; float:right" /><br />
<p><br />
Terpene biosynthesis in plants is part of larger pathways that metabolize isoprenoid intermediates. Genes encoding for enzymes known as synthases catalyze the terminal step in these pathways, from a precursor (commonly farnesyl pyrophosphate (FPP) or garnyl pyrophosphate (GPP)) to the final terpene product. As it happens, two well established and genetically manipulable model organisms- the bacterium <i> Escherichia coli </i> and baker's yeast <i> Saccharomyces cerevisiae</i>- produce moderate amounts of GPP and FPP as part of their endogenous non-mevalonate pathway (MEP) and mevalonate pathway (MEV) respectively<sup>3</sup>. All that is required for either of these organisms to begin producing terepenes is to introduce that single synthase gene.<br />
</p><br />
<p><br />
Yeast initially was our main target for a few advantages it appeared to have as a production platform. First, the MEV pathway is found in all eukaryotes including plants and fungi, so better yield were expected by choosing it over the MEP pathway in prokaryotes<sup>4</sup>. Second, it would be possible to physically integrate our gene into one of the yeast's chromosomes by using homologous recombination. An inducible promoter could be included to further increase production. Third, as a diploid the yeast could be made homozygous for the terpene gene. We soon found out that no exiting vector had all of the features we would want. Therefore, we designed our own new plasmid vector, pVU140006, that contained a number of important features and advances over previous plasmids for this purpose. See our parts page for more information on what special features we included and their relevance to the project<br />
</p><br />
<br />
</tr><br />
<tr><br />
<td><h3>Methods</h3><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/8/82/VU_experiment_1_diagram.png" align=left alt="First few experiments" width="300" style="padding-bottom:0.5em; float:right" /><br />
<p><br />
Our project had several co-dependent sub-project that were all worked on in parallel. These can roughly be divided into two categories: the first involving work on our synthase genes and the second involving the construction of a new, specially designed plasmid vector. We tried two different team structures over the year to see which would give the best results. For the Spring, we had the original idea of dividing members into independent groups, each working on a specific terpene. Each group was headed by a single group manager who would teach 4-5 new members the protocol that was to be preformed and then supervise that the work was carried out correctly. On occasion either the group managers or the organization president or wetware director would also given lessons to teach members about the techniques and theory involved at each step. All group managers were in turn trained by either the president or wetware director, both of whom had come with the experiment, acquired all the necessary primers and reagents, wrote up the protocol, and had preformed it prior to any group-phase work for the purposes of troubleshooting and predicting where issues may come up. The president or wetware director also helped the group manager in being present during all experiments for answering questions, preparing materials, and other forms of assistance. <br />
</p><br />
<p><br />
Each group first planted seeds under the appropriate soil, humidity, and temperature conditions at the Vanderbilt Greenhouse. Once the majority of these grew into saplings with green leaves, Samples were flash frozen in liquid nitrogen in preparation for a genomic DNA extraction. After the extractions, nanodrop concentration readings and agarose gels confirmed the presence of high molecular weight genomic DNA. Groups then ran a PCR on their genomic DNA with primers specific to their synthase gene. More gels were run to check for PCR product. At this point, the semester was coming to an end, so groups were disbanded before most managed to isolate their synthase gene. Over the summer, the president and wetware director continued troubleshooting those genes which were not amplifying and eventually got each to the point where consistent PCR product bands were produced. </p><br />
<br />
<img src="https://static.igem.org/mediawiki/parts/f/fd/VU_experiment_2_diagram.png" align=right alt="First few experiments" width="300" style="padding-bottom:0.5em; float:right" /><br />
<br />
<p><br />
Once we had clear banding patterns, it became clear that the number of introns in each of our genes (a variable which was unknown since most of the plants we worked with have not had their genomes sequenced) was too great for cloning and expression to be practical. Therefore, as soon as the fall semester began, we shifted strategy to isolating RNA from our plants. This RNA could be converted to cDNA by reverse transcription, which would eliminate the issue with introns we were having. Several of our greenhouse plants were no longer available, so we reduced the number of target terpenes we were focusing on. After extracting RNA and running an RT-PCR, several samples produced bands that corresponded roughly to where the synthase gene should be. These were gel extracted. Because almost every synthase gene had restriction sites in them that prevented them from being RFC10 compatible, we ligated the genes in pUC19 for site directed mutagenesis. After that, a second processing step would have been necessary to add the correct restriction sites to each gene to allow them to be integrated into pSB1C3 as a biobrick. In the interest of time, we synthesized a codon-optimized santalene synthase gene in order to skip these RFC10 processing steps, even though we had already successfully reverse transcribed cDNA of the santalene synthase gene.<br />
</p><br />
<p><br />
In the spring concurrent to work by the terpene groups, work began on plasmid construction. This was preformed by the president, wetware director, and a handful of others rather than in group format. Each gene cassette for our final plasmid was first identified in an existing, readily available plasmid. All of these cassettes were extracted by PCR using those plasmids as templates. Overlap extension PCR was then done on the gel-purified product to add restriction sites and homology regions for the purposes of eventually combining all of the cassettes together into a single plasmid. By the end of the summer, only one final fragment remained to be inserted to complete the intermediate plasmid pVU14004. Upon the successful creation of pVU14004, several restriction enzyme sites had to be removed by site directed mutagensis in order to make the plasmid RFC10 compatible. <br />
</p><br />
<br />
<h3> Results and Directions </h3><br />
<p><br />
Several factors contributed to the difficulty we experienced during the final phase of the project. First, member engagement suffered a significant decline between the spring and fall semesters, to the point where only a small handful of people were left to preform all experiments. Second, the late realization that we had to change our cloning strategy to modified cDNA inserts effectively meant we had to start anew in late August despite having what was a good head start when we began in early March. Third, the RFC10 requirements added a substantial dimension of difficulty to the project since all of our starting material (both the extracted gene cassettes for plasmid construction and the synthase genes) contained multiple sites that made them incompatible with the biobrick standard. Nevertheless, our team accomplished an enormous amount during our first year in competition. </p><br />
<br><br />
<table width="90%"><br />
<tr><br />
<td> <i>Terpene </i> </td><br />
<td> <i>Plant Genomic DNA Successfully Extracted</i> </td><br />
<td> <i>Synthase Gene Successfully PCR Isolated </i> </td><br />
<td> <i>Plant RNA Successfully Extracted</i> </td><br />
<td> <i>Synthase gene Successfully Reverse Transcribed</i> </td><br />
<td> <i>Terpene Successfully Produced in E. coli or yeast <i> </td><br />
<tr><br />
<td> <b>Cadinene</b> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td> <br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td> <br />
</tr><br />
<tr><br />
<td> <b>Carene</b> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td> <br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td> <br />
</tr><br />
<tr><br />
<td> <b>Humelene</b> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td> <br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td> <br />
</tr><br />
<tr><br />
<td> <b>Myrcene</b> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td> <br />
</tr><br />
<tr><br />
<td> <b>(R)-Linalool</b> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td> <br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td> <br />
</tr><br />
<tr><br />
<td> <b>(S)-Linalool</b> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td> <br />
</tr><br />
<tr><br />
<td> <b>Sabinene</b> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5b/Yellow_square.png" width="50"> </td> <br />
</tr><br />
<tr><br />
<td> <b>Santalene</b> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5b/Yellow_square.png" width="50"> </td><br />
</tr><br />
<tr><br />
<td> <b>Zingiberene</b> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="50"> </td><br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td> <br />
<td> <img src="https://static.igem.org/mediawiki/parts/5/5f/Red_X.png" width="50"> </td> <br />
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<img src="https://static.igem.org/mediawiki/parts/2/28/Green_check_mark.png" width="100" align="right"><br />
<p><br />
We successfully constructed pVU14004, which contains all of the cassettes needed to have all of functionality we had intended. However, this form of the plasmid has XbaI and EcoRI sites that make it incompatible with RFC10. These sites were successfully changed to missense mutations by site directed mutagenesis. The final step required to make pVU14006 is changing the multiple cloning site (MCS) to have the biobrick prefix and suffix. In its current form, the plasmid has a functional MCS but it does not yet have the specific order of EcoRI, XbaI, SpeI, and PstI sites required by RFC10. <br />
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<p><br />
For a listing of all the medal requirements we successfully fulfilled over the course of our project, please visit <a href="https://2014.igem.org/Team:Vanderbilt/Project/Requirements"style="color:#000000"> <u> this page </u></a>.<br />
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<h3> Collaborations </h3><br />
<img src="https://static.igem.org/mediawiki/parts/6/64/Ravenwood_high_school.jpg" width="300" align="left"><br />
<p><br />
In addition to our own wetware project, our team led fruitful collaborations with a total of three other iGEM teams. First, we played a major role in assisting Vanderbilt's microfluidic division with the biological aspect of their project. We prepared the biobrick parts they tested in their microfluidic device, including transforming the E. coli they used to study their quorum-sensing fluorescent oscillator circuit. Second, we provided feedback to Vanderbilt's software division about their own project involving a program to aid in the manipulation of genetic sequences. We used the program as if it were for a real project and gave them suggestions on how to make their program easier to use and more useful to biologists.<br />
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<p> <br />
Last but not least, we provided significant assistance and guidance to the Ravenwood Raptors high school iGEM team. In a series of conversations with Dr. Amanda Benson, we planned a smaller scale version of our own project that involved only a single terpene synthase gene. After visiting the high school and presenting our idea, the students voted to choose it for their project. We also provided the primers and sage genomic DNA template they used in their experiments. <br />
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<b> References: </b></br><br />
<i>1. USDA Industrial Uses Reports. Essential Oils Widely Used in Flavors and Fragrances. September 1995. <br><br />
2. Ajikumar PK, Tyo K, Carlsen S, Mucha O, Phon TH, Stephanopoulos G. Terpenoids: opportunities for biosynthesis of natural product drugs using engineered microorganisms. Mol Pharm. 2008;5(2):167-90. <br><br />
3. Dudareva N, Klempien A, Muhlemann JK, Kaplan I. Biosynthesis, function and metabolic engineering of plant volatile organic compounds. New Phytol. 2013;198(1):16-32. <br><br />
4.Martin VJ, Pitera DJ, Withers ST, Newman JD, Keasling JD. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol. 2003;21(7):796-802. <br><br />
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