Team:Vanderbilt/Parts

From 2014.igem.org

(Difference between revisions)
(Prototype team page)
 
(38 intermediate revisions not shown)
Line 1: Line 1:
{{CSS/Main}}
{{CSS/Main}}
-
 
<html>
<html>
<style type="text/css">
<style type="text/css">
-
#groupparts {text-align: center; margin-left: auto; margin-right: auto;}
+
body {
 +
    position: relative;
 +
    width: 850px //100%;
 +
    margin: 0;
 +
    padding: 0;
 +
    padding-bottom: 10px;
 +
    background:url(https://static.igem.org/mediawiki/parts/8/86/VU_Campus_photo_10.JPG);
 +
    background-repeat: no-repeat;
 +
    background-attachment: fixed;
 +
    background-size:100% auto;
 +
    font-family: Georgia, Times, "Times New Roman", serif;
 +
}
 +
 
 +
 
 +
table {
 +
cellpadding: 10;
 +
cellspacing:5;
 +
width: 75%;
 +
margin-left: auto;  
 +
margin-right: auto;
 +
background-color: rgba(204,153,0,0.8);
 +
border-radius: 8px;
 +
height: 60px;
 +
colspan=2;
 +
padding:5px;
 +
}
 +
 
 +
p {
 +
font-size: 1.25em;
 +
}
 +
 
 +
.firstHeading { display: none;}
 +
.printfooter { display: none; }
</style>
</style>
 +
 +
<center><img src="https://static.igem.org/mediawiki/parts/7/72/VU_iGem_Logo_%28Transparent.png" align="middle" width="500px"></center>
<!--main content -->
<!--main content -->
-
<table width="70%" align="center">
+
<table width="70%" align="center" margin-bottom: "3cm"; style="border:4px solid black;" style="margin: 1em auto 1em auto;">
-
 
+
-
 
+
-
<!--welcome box -->
+
-
<tr>
+
-
<td style="border:1px solid black;" colspan="3" align="center" height="150px" bgColor=#FF404B>
+
-
<h1 >WELCOME TO iGEM 2014! </h1>
+
-
<p>Your team has been approved and you are ready to start the iGEM season!
+
-
<br>On this page you can document your project, introduce your team members, document your progress <br> and share your iGEM experience with the rest of the world! </p>
+
-
<br>
+
-
<p style="color:#E7E7E7"> <a href="https://2014.igem.org/wiki/index.php?title=Team:Vanderbilt/Parts&action=edit"style="color:#FFFFFF"> Click here  to edit this page!</a> </p>
+
-
</td>
+
-
</tr>
+
-
<tr> <td colspan="3"  height="5px"> </td></tr>
 
-
<!-- end welcome box -->
 
<tr>  
<tr>  
Line 28: Line 47:
<td align="center" colspan="3">
<td align="center" colspan="3">
-
<table  width="100%">
+
<table  width="100%" padding-bottom: "15">
 +
<border-width=1px >
<tr heigth="15px"></tr>
<tr heigth="15px"></tr>
<tr heigth="75px">  
<tr heigth="75px">  
 +
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000> 
 +
<a href="https://2014.igem.org/Team:Vanderbilt"style="color:#CC9900">Home<br>
 +
<img src="https://static.igem.org/mediawiki/parts/c/cc/VU_vumc_home.jpg" width="150px"> </td> </a>
-
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7>
+
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000>  
-
<a href="https://2014.igem.org/Team:Vanderbilt"style="color:#000000">Home </a> </td>
+
<a href="https://2014.igem.org/Team:Vanderbilt/Team"style="color:#CC9900">Team</br>
 +
<img src="https://static.igem.org/mediawiki/parts/d/d3/Lab_work_banner_image.JPG" width="120px"> </td> </a>  
-
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7>  
+
<td style="border:1px solid black" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000>
-
<a href="https://2014.igem.org/Team:Vanderbilt/Team"style="color:#000000"> Team </a> </td>
+
<a href="https://2014.igem.org/Team:Vanderbilt/Project"style="color:#CC9900">Project<br>
 +
<img src="https://static.igem.org/mediawiki/parts/0/09/VU_Yeast_tubes_front.JPG" width="120px"> </td></a>
-
<td style="border:1px solid black;" align="center"  height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7>  
+
<td style="border:1px solid black;" align="center"  height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000>  
-
<a href="https://igem.org/Team.cgi?year=2014&team_name=Vanderbilt"style="color:#000000"> Official Team Profile </a></td>
+
<a href="https://2014.igem.org/Team:Vanderbilt/Parts"style="color:#CC9900">Parts</br>
 +
<img src="https://static.igem.org/mediawiki/parts/8/8b/VU_transparent_pVU.gif" width="150px"> </td></a>
-
<td style="border:1px solid black" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7>   
+
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000>   
-
<a href="https://2014.igem.org/Team:Vanderbilt/Project"style="color:#000000"> Project</a></td>
+
<a href="https://2014.igem.org/Team:Vanderbilt/Notebook"style="color:#CC9900">Notebook<br>
 +
<img src="https://static.igem.org/mediawiki/parts/c/ca/VU_sample_Lab_Journal_Page.JPG" width="90px"></td></a>  
-
<td style="border:1px solid black;" align="center"  height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7>  
+
<td style="border:1px solid black;" align="center"  height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000>  
-
<a href="https://2014.igem.org/Team:Vanderbilt/Parts"style="color:#000000"> Parts</a></td>
+
<a href="https://2014.igem.org/Team:Vanderbilt/Safety"style=" color:#CC9900">Safety</br>
 +
<img src="https://static.igem.org/mediawiki/parts/e/eb/Safety_logo.gif" width="90px"></td></a>
-
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7>  
+
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#000000'" bgColor=#000000>  
-
<a href="https://2014.igem.org/Team:Vanderbilt/Modeling"style="color:#000000"> Modeling</a></td>
+
<a href="https://2014.igem.org/Team:Vanderbilt/Attributions"style="color:#CC9900">Attributions<br>
-
 
+
<img src="https://static.igem.org/mediawiki/parts/0/0e/Attributions_page_clipart.jpg" width="130px"></td></a>
-
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7> 
+
-
<a href="https://2014.igem.org/Team:Vanderbilt/Notebook"style="color:#000000"> Notebook</a></td>
+
-
 
+
-
<td style="border:1px solid black;" align="center"  height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7>
+
-
<a href="https://2014.igem.org/Team:Vanderbilt/Safety"style=" color:#000000"> Safety </a></td>
+
-
 
+
-
<td style="border:1px solid black;" align="center"  height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7>
+
-
<a href="https://2014.igem.org/Team:Vanderbilt/Attributions"style="color:#000000"> Attributions </a></td>
+
Line 65: Line 85:
</table>
</table>
-
<!--end navigation menu -->
 
</tr>
</tr>
-
 
-
 
</tr>
</tr>
-
 
-
 
-
 
-
 
-
 
</td>
</td>
-
<tr> <td colspan="3" height="15px"> </td></tr>
+
<!--main content -->
-
<tr><td bgColor="#e7e7e7" colspan="3" height="1px"> </tr>
+
<table width="70%" align="center">
-
<tr> <td colspan="3"  height="5px"> </td></tr>
+
<tr><td>
 +
<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" />
 +
<h3> BBa_K1322231- Optimized Santalene Synthase</h3>
 +
<p>
 +
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.
 +
</p>
-
 
-
<!--Parts Submitted to the Registry  -->
 
-
<tr><td > <h3> Parts Submitted to the Registry </h3></td>
 
-
<td ></td >
 
-
<td > <h3>What information do I need to start putting my parts on the Registry? </h3></td>
 
-
</tr>
 
-
<tr>
 
-
<td width="45%"  valign="top">
 
<p>
<p>
-
An important aspect of the iGEM competition is the use and creation of standard  biological parts. Each team will make new parts during iGEM and will submit them to the <a href="http://partsregistry.org"> Registry of Standard Biological Parts</a>. The iGEM software provides an easy way to present the parts your team has created. The "groupparts" tag will generate a table with all of the parts that your team adds to your team sandbox. 
+
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.
 +
</p>
<p>
<p>
-
<strong>Note that if you want to document a part you need to document it on the <a href="http://partsregistry.org Registry"> Registry</a>, not on your team wiki.</strong> Future teams and other users and are much more likely to find parts on the Registry than on your team wiki.  
+
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).
</p>
</p>
<p>
<p>
-
Remember that the goal of proper part documentation is to describe and define a part, so that it can be used without a need to refer to the primary literature. Registry users in future years should be able to read your documentation and be able to use the part successfully. Also, you should provide proper references to acknowledge previous authors and to provide for users who wish to know more.
+
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.
</p>
</p>
 +
<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" />
-
<h3>When should you put parts into the Registry?</h3>
+
<h3> pVU14006 </h3>
-
 
+
<p>
<p>
-
As soon as possible! We encourage teams to start completing documentation for their parts on the Registry as soon as you have it available. The sooner you put up your parts, the better recall you will have of all details surrounding your parts. Remember you don't need to send us the DNA to create an entry for a part on the Registry. However, you must send us the sample/DNA before the Jamboree. Only parts for which you have sent us samples/DNA are eligible for awards and medal requirements.  
+
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.  
</p>
</p>
-
</td>
 
-
 
-
<td > </td>
 
-
<td width="45%" valign="top">
 
<p>
<p>
-
The information needed to initially create a part on the Registry is:
+
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.
</p>
</p>
-
<ol>
 
-
 
-
<li>Part Name</li>
 
-
<li>Part type</li>
 
-
<li>Creator</li>
 
-
<li>Sequence</li>
 
-
<li>Short Description (60 characters on what the DNA does)</li>
 
-
<li>Long Description (Longer description of what the DNA does)</li>
 
-
<li>Design considerations</li>
 
-
</ol>
 
 +
<h3> BBa_K1322001- All-RFC Compatible Fluorescent Oscillator</h3>
<p>
<p>
-
We encourage you to put up <em>much more</em> information as you gather it over the summer. If you have images, plots, characterization data and other information, please also put it up on the part page. Check out part <a href="http://parts.igem.org/Part:BBa_K404003">BBa_K404003</a> for an excellent example of a highly characterized part.  
+
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.  
</p>
</p>
 +
<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" />
 +
 +
<h3> Sabinene Synthase </h3>
<p>
<p>
-
You can add parts to the Registry at our <a href="http://parts.igem.org/Add_a_Part_to_the_Registry"> Add a Part to the Registry</a> link.
+
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.
</p>
</p>
-
</td>
 
-
</tr>
 
-
 
+
<br>
-
<tr> <td colspan="3"  height="15px"> </td></tr>
+
<h3>References:</h3>
-
 
+
<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>
-
<tr><td colspan="3" > <h3> Parts Table</h3></td></tr>
+
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
-
 
+
Koch et al: Inhibitory effect of essential oils against herpes simplex virus type 2. Phytomedicine 2008;15(1-2):71-8. <br>
-
 
+
Rebecca Tirabassi, How to identify supercoils, nicks and circles in plasmid preps. Bitesizebio. October 8, 2014.</i><p>
-
<tr><td width="45%" colspan="3"  valign="top">
+
-
Any parts your team has created will appear in this table below:</td></tr>
+
-
 
+
-
</table>
+
-
</html>
+
-
 
+
-
<groupparts>iGEM013 Vanderbilt</groupparts>
+

Latest revision as of 02:22, 9 February 2015

Home
Team
Project
Parts
Notebook
Safety
Attributions

BBa_K1322231- Optimized Santalene Synthase

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.

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.

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).

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 S. cerevisiae. 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.

pVU14006

pVU14006

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.

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.

BBa_K1322001- All-RFC Compatible Fluorescent Oscillator

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.

Sabinene Synthase

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.


References:

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
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 Koch et al: Inhibitory effect of essential oils against herpes simplex virus type 2. Phytomedicine 2008;15(1-2):71-8.
Rebecca Tirabassi, How to identify supercoils, nicks and circles in plasmid preps. Bitesizebio. October 8, 2014.