Team:Hong Kong HKUST/Project/module3

From 2013.igem.org

(Difference between revisions)
 
(54 intermediate revisions not shown)
Line 8: Line 8:
$(this).find("ul").stop().fadeToggle(10);
$(this).find("ul").stop().fadeToggle(10);
});
});
 +
});
 +
</script>
 +
<script>
 +
$(document).ready(function(){
 +
 +
// hide #back-top first
 +
$("#back-top").hide();
 +
 +
// fade in #back-top
 +
$(function () {
 +
$(window).scroll(function () {
 +
if ($(this).scrollTop() > 100) {
 +
$('#back-top').fadeIn();
 +
} else {
 +
$('#back-top').fadeOut();
 +
}
 +
});
 +
 +
// scroll body to 0px on click
 +
$('#back-top a').click(function () {
 +
$('body,html').animate({
 +
scrollTop: 0
 +
}, 800);
 +
return false;
 +
});
 +
});
 +
});
});
</script>   
</script>   
Line 42: Line 69:
{visibility:hidden;}
{visibility:hidden;}
#globalWrapper
#globalWrapper
-
{position: relative;font-size:127%;width:100%;margin:0;padding:0;padding-bottom:10px;background-color:#494042;}
+
{position: relative;font-size:127%;width:100%;margin:0;padding:0;padding-bottom:10px;background-color:#494042;height:1500px;}
#content
#content
-
{width: 1000px;border:none;background-color:#ffffff;padding:0px;margin-top:0px;line-height: 1.5em;color: black;}
+
{width: 995px;border:none;background-color:#ffffff;height:1450px;padding:0px;margin-top:0px;line-height: 1.5em;color: black;}
#top-section
#top-section
{height:2px;border:none;background-color:#494042;}
{height:2px;border:none;background-color:#494042;}
#catlinks
#catlinks
-
{border-color:#494042;background-color:#494042;}
+
{display:none;}
#footer-box
#footer-box
{display:none}
{display:none}
Line 55: Line 82:
#cover
#cover
{position:absolute;top:0px;height:168px;width:100%;background:#494042;z-index:-1;}
{position:absolute;top:0px;height:168px;width:100%;background:#494042;z-index:-1;}
-
body, div, dl, dt, dd, ul, ol, li, h1, h2, h3, h4, h5, h6, pre, form, fieldset, p, blockquote, th, td {
+
body, div, dl, dt, dd, ul, ol, li, h1, h4, h6, pre, form, fieldset, p, blockquote, th, td {
margin: 0;
margin: 0;
padding: 0;
padding: 0;
Line 93: Line 120:
{
{
float:left;
float:left;
-
width:16.6667%;
+
width:16.667%;
}
}
ul.first>li>a:link,ul.first>li>a:visited
ul.first>li>a:link,ul.first>li>a:visited
Line 140: Line 167:
top:141px;
top:141px;
width:100%;
width:100%;
 +
z-index:1;
}
}
-
ul.side-nav {
+
#back-top {
 +
position: fixed;
 +
top: 10%;
 +
right: 2%;
 +
z-index:16;
 +
}
 +
#back-top a {
 +
width: 108px;
 +
display: block;
 +
text-align: center;
 +
font: 11px/100% Arial, Helvetica, sans-serif;
 +
text-transform: uppercase;
 +
text-decoration: none;
 +
color: #bbb;
 +
/* background color transition */
 +
-webkit-transition: 1s;
 +
-moz-transition: 1s;
 +
transition: 1s;
 +
}
 +
#back-top a:hover {
 +
color: #ffffff;
 +
}
 +
 
 +
#back-top span {
 +
width: 65px;
 +
height: 60px;
 +
display: block;
 +
margin-bottom: 7px;
 +
background: #ddd url(up-arrow.png) no-repeat center center;
 +
/* rounded corners */
 +
-webkit-border-radius: 15px;
 +
-moz-border-radius: 15px;
 +
border-radius: 15px;
 +
/* background color transition */
 +
-webkit-transition: 1s;
 +
-moz-transition: 1s;
 +
transition: 1s;
 +
}
 +
#back-top a:hover span {
 +
background-color: #777;margin-top:10px;
 +
}
 +
 
 +
ul.side-nav, ul.side-nav1 {
display: block;
display: block;
list-style: none;
list-style: none;
margin: 0;
margin: 0;
-
padding: 17px 0;
+
padding: 17px 0;font-family:"Trebuchet MS", Helvetica, sans-serif;
}
}
ul.side-nav h6 {
ul.side-nav h6 {
margin: 47px 0px 0px;
margin: 47px 0px 0px;
}
}
-
ul.side-nav li.divider {
+
ul.side-nav1 h6 {
 +
margin: 0px 0px 0px;
 +
}
 +
ul.side-nav li.divider, ul.side-nav1 li.divider {
border-top: 1px solid #ddd;
border-top: 1px solid #ddd;
}
}
 +
.two, .row .two {
.two, .row .two {
width: 16.66667%;
width: 16.66667%;
Line 167: Line 241:
width: 83.33333%;
width: 83.33333%;
top:192px;
top:192px;
-
z-index:-2;
+
 
}
}
.column, .columns {
.column, .columns {
Line 178: Line 252:
width: 100%;
width: 100%;
}
}
-
h1, h2, h3, h4, h5, h6 {
+
h1, h2, h4, h5, h6 {
-
font-family: 'Bitter', serif;
+
font-family:"Trebuchet MS", Helvetica, sans-serif;
font-weight: normal;
font-weight: normal;
color: #000;
color: #000;
border-bottom: none;
border-bottom: none;
 +
 +
 +
h3{font-size:18px;font-family:"Trebuchet MS", Helvetica, sans-serif;}
 +
h5{margin-left:15px;}
 +
}
}
div.team-bios-container div.row {
div.team-bios-container div.row {
Line 205: Line 284:
}
}
.nine, .row .nine {
.nine, .row .nine {
-
width: 75%;
+
width: 100%;font-family:"Trebuchet MS", Helvetica, sans-serif;
}
}
[class*="column"] + [class*="column"]:last-child {
[class*="column"] + [class*="column"]:last-child {
Line 213: Line 292:
width: 25%;
width: 25%;
}
}
-
</style>
+
#construct{margin-left:30px;}
 +
ul li ul, ul li ol {
 +
margin-left: 20px;
 +
margin-bottom: 0;}
 +
#iGEM_Logo{
 +
width:100px;
 +
height:80px;
 +
position:absolute;
 +
right:10px;
 +
top:60px;
 +
z-index:+15;
 +
}
 +
#hkust_Logo{
 +
width:60px;
 +
height:80px;
 +
position:absolute;
 +
right:110px;
 +
top:60px;
 +
z-index:+15;
 +
}
 +
 +
</style>
 +
</head>
<body>
<body>
 +
 +
          <a href="https://2013.igem.org/Main_Page"><img id="iGEM_Logo" src="https://static.igem.org/mediawiki/2013/4/46/Igem_qgem_logo.png"></a>
 +
         
 +
 +
<a href="http://www.ust.hk/eng/index.htm"><img id="hkust_Logo" src="https://static.igem.org/mediawiki/2013/5/55/Hkust_logo.gif"></a>
 +
 +
<p id="back-top" style="display:none;">
 +
<a href="#top"><span><img src="http://515alive.com/theme/img/up-arrow.png" style="width:90%;"><br><br>BACK TO TOP</span></a>
 +
</p>
<a href=https://2013.igem.org/Team:Hong_Kong_HKUST><center><div id="kepala" style="height:121px;width:100%;"><img src="https://static.igem.org/mediawiki/igem.org/c/c7/BANNER1_%281%29.png" style="height:121px;width:100%;align:middle;"></div></center></a>
<a href=https://2013.igem.org/Team:Hong_Kong_HKUST><center><div id="kepala" style="height:121px;width:100%;"><img src="https://static.igem.org/mediawiki/igem.org/c/c7/BANNER1_%281%29.png" style="height:121px;width:100%;align:middle;"></div></center></a>
<div id="cover"></div>
<div id="cover"></div>
Line 228: Line 338:
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/advisors">Advisors</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/advisors">Advisors</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/instructors">Instructors</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/instructors">Instructors</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/attribution">Attribution</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/acknowledge">Acknowledgement</a></li>
</ul>
</ul>
</li>
</li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/project">Project</a>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Project">Project</a>
<ul>
<ul>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/abstract">Abstract</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/abstract">Abstract</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/modelling">Modelling</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/modules">Modules Description</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/characterization">Characterization</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/data">Data Page</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Parts">Parts</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Parts">Parts</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/datapage">Data Page</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/characterization">Characterization</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/results">Results</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/results">Result</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/future">Future Work</a></li>
</ul>
</ul>
</li>
</li>
Line 244: Line 357:
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Wetlab">Wetlab</a>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Wetlab">Wetlab</a>
<ul>
<ul>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook">notebook</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook">Notebook</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/protocols">protocols</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/protocols">Protocols</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/safety">safety</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/safety">Safety</a></li>
 +
 
</ul>
</ul>
</li>
</li>
Line 259: Line 373:
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp">Human Practice</a>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp">Human Practice</a>
<ul>
<ul>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/Interviews">Interviews</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/cp">Country Profile</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/Presentation">Presentation</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/blog">Blog</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/Videos">Videos</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/interview">Interviews</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/Article">Article</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/article/genet">Article</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/video">Videos</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/presentation">Presentations</a></li>
</ul>
</ul>
</li>
</li>
</ul>
</ul>
 +
</div>
</div>
-
</div>
+
      <div class="row">
-
<div class="row">
+
<div class="two columns">
<div class="two columns">
-
<ul class="side-nav">
+
<br>
 +
<ul class="side-nav">
<li>
<li>
-
<h6>Module Three</h6>
+
<h6>Modules</h6>
</li>
</li>
<li class="divider"></li>
<li class="divider"></li>
-
<li>
+
<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Project/module4">Glyoxylate Shunt</a>
-
<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Wetlab">Introduction</a>
+
</li>
</li>
 +
<li>
 +
Protein Trafficking
 +
<ul><li>
 +
<li>
<li>
-
<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Wetlab">Mechanism of MLS</a>
+
<a href=#1>Overview</a>
</li>
</li>
-
 
<li>
<li>
-
<a href="https://2012.igem.org/Team:Cornell/project/drylab/components">Parts Submission</a>
+
<a href=#2>Biology of Mitochondrial Leader Sequence (MLS)</a>
</li>
</li>
 +
<li>
<li>
-
<a href="https://2012.igem.org/Team:Cornell/project/drylab/status">Characterization</a>
+
<a href=#3>Reference</a>
 +
                                               
 +
</li>
 +
</ul>
 +
</li>
 +
<li>
 +
<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Project/module2">FA Sensing Mechanism</a>
 +
 
</li>
</li>
-
+
</ul>
-
</ul>
+
 
</div>
</div>
<div class="ten columns team-bios-container">
<div class="ten columns team-bios-container">
Line 299: Line 427:
</div>
</div>
<div class="row">
<div class="row">
-
<div class="nine columns">
+
<div class="nine columns"><p id="1"></p>
-
<h3>Introduction</h3>
+
<h3>Overview</h3>
 +
In our project, we introduced bacterial glyoxylate enzymes into mammalian cells to create alternative metabolic pathway. However, unlike their native environment in bacteria, the two enzymes needed to find their way through the high compartmentalized system in order to reach the citric acid cycle where they could act on.
<br>
<br>
-
In our project ,we will introduce bacterial enzymes to mammalian cell to modify metabolic pathway. However, unlike bacteria, citric acid cycle in mammalian cells is compartmentalized in mitochondria. The ACE proteins should be targeted to mitochondria for their functionality. To do so, we fused ACE enzymes with Mitochondrial Leader Sequence (MLS).
+
To guide the glyoxylate enzymes into the mitochondria where mammalian citric acid cycle resides, we produced recombinant glyoxylate enzymes by attaching the Mitochondrial Leader Sequence (MLS) to their N-termini.
-
<br>
+
<br>
 +
We have also constructed the MLS in its own into standard BioBricks (<a href="http://parts.igem.org/Part:BBa_K1119000">BBa_K1119000</a> & <a href="http://parts.igem.org/Part:BBa_K1119001">BBa_K1119001</a>), and we quantitatively characterized their behavior using GFP reporter.
</div>
</div>
-
<div class="three columns">
+
</div>
-
+
<div class="row">
 +
<div class="nine columns"><p id="2"></p>
 +
<h3>Biology of Mitochondrial Leader Sequence (MLS)</h3>
 +
<p>In eukaryotes, the signal sequence guides the translocation of the newly synthesized peptide.</p>
 +
<p>The story starts with the MLS attached to the N-terminus of the protein of interest. Once the protein of interest is synthesized, this preprotein would remain unfolded by associating with chaperons. The preprotein will stay in the cytosol until the MLS gets recognized by receptor of the TOM complex on the outer mitochondrial membrane. Binding of the MLS to the receptor will trigger the feeding of the peptide through the translocation channel. Afterwards, the MLS will then be handed over to a TIM complex which sits on the inner membrane, which will then open up the channels on the inner membrane and allow the peptide to pass through. Once the peptide is through the double membranes, mitochondrial chaperone will be involved in pulling the peptide into the mitochondria and refold the protein. Lastly, the MLS will be cleaved by signal peptidase and dissociate from the transported peptide. (Alberts, 2002) <br>For the MLS we used, four additional amino acid residues (Ile-His-Ser-Leu) will be left at the N-terminus of the protein after the cleavage.(Invitrogen, 2012)</p>
 +
<img src="https://static.igem.org/mediawiki/2013/8/87/MLS_mechanism.png" >
</div>
</div>
</div>
</div>
<div class="row">
<div class="row">
-
<div class="three columns">
+
<div class="nine columns"><p id="3"></p>
-
</div>
+
<div class="nine columns"><p id="5"></p>
-
<div class="nine columns">
+
<h3>Reference</h3>
-
<h3>Mechanism of Mitochondrial Leader Sequence</h3>
+
Alberts, B., Bray, D., Hopkin, K., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2010). <i>Essential cell biology.</i> (3rd ed., p. 505). UK: Garland Science.<br><br>
-
<br>
+
pCMV/<i>myc</i>/mito Invitrogen.(2012).pShooter™ Vector(pCMV/<i>myc</i> vectors).Retrieved from http://tools.lifetechnologies.com/content/sfs/manuals/pshooter_pcmv_man.pdf <br><br>
-
MLS will be attached to the N-terminal of enzyme, and bind to the receptor protein on mitochondrial membrane, and diffuse to contact site where inner membrane & outer membrane fuse, then bring the ACE enzyme into mitochondria. Afterward, it will be cleaved , leaving the enzyme in mitochondria.
+
-
 
+
Alberts, B. (2002). <i>Molecular biology of the cell.</i> (4th ed., pp. 1050-1061). New York:Garland Science.
-
</div>
+
-
</div>
+
-
<div class="row">
+
-
<div class="nine columns">
+
-
<h3>Part Submission</h3>
+
<br>
<br>
-
The MLS was cloned from a commercial plasmid, pCMV/myc/mito (Invitrogen) by PCR. For the MLS biobrick, we are going to submit the MLS biobrick RFC 10 and RFC 25 , the Freiburg format which allow protein fusion , to facilitate other team to fuse the MLS with other protein for purpose of introducing other protein into mitochondria.
+
</div>
-
The MLS is sequenced and is confirmed to be free from illegal site. It is ligated with pSB1C3 in biobrick.
+
-
 
+
-
</div>
+
-
<div class="three columns">
+
-
+
-
</div>
+
-
</div>
+
-
<div class="row">
+
-
+
</div>
</div>
</div>
</div>
-
</div>
+
 
</body>
</body>
</html>
</html>

Latest revision as of 12:42, 28 October 2013

Protein Trafficking

Overview

In our project, we introduced bacterial glyoxylate enzymes into mammalian cells to create alternative metabolic pathway. However, unlike their native environment in bacteria, the two enzymes needed to find their way through the high compartmentalized system in order to reach the citric acid cycle where they could act on.
To guide the glyoxylate enzymes into the mitochondria where mammalian citric acid cycle resides, we produced recombinant glyoxylate enzymes by attaching the Mitochondrial Leader Sequence (MLS) to their N-termini.
We have also constructed the MLS in its own into standard BioBricks (BBa_K1119000 & BBa_K1119001), and we quantitatively characterized their behavior using GFP reporter.

Biology of Mitochondrial Leader Sequence (MLS)

In eukaryotes, the signal sequence guides the translocation of the newly synthesized peptide.

The story starts with the MLS attached to the N-terminus of the protein of interest. Once the protein of interest is synthesized, this preprotein would remain unfolded by associating with chaperons. The preprotein will stay in the cytosol until the MLS gets recognized by receptor of the TOM complex on the outer mitochondrial membrane. Binding of the MLS to the receptor will trigger the feeding of the peptide through the translocation channel. Afterwards, the MLS will then be handed over to a TIM complex which sits on the inner membrane, which will then open up the channels on the inner membrane and allow the peptide to pass through. Once the peptide is through the double membranes, mitochondrial chaperone will be involved in pulling the peptide into the mitochondria and refold the protein. Lastly, the MLS will be cleaved by signal peptidase and dissociate from the transported peptide. (Alberts, 2002)
For the MLS we used, four additional amino acid residues (Ile-His-Ser-Leu) will be left at the N-terminus of the protein after the cleavage.(Invitrogen, 2012)

Reference

Alberts, B., Bray, D., Hopkin, K., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2010). Essential cell biology. (3rd ed., p. 505). UK: Garland Science.

pCMV/myc/mito Invitrogen.(2012).pShooter™ Vector(pCMV/myc vectors).Retrieved from http://tools.lifetechnologies.com/content/sfs/manuals/pshooter_pcmv_man.pdf

Alberts, B. (2002). Molecular biology of the cell. (4th ed., pp. 1050-1061). New York:Garland Science.

Retrieved from "http://2013.igem.org/Team:Hong_Kong_HKUST/Project/module3"