Team:UC Davis/Project/Protein Engineering

From 2012.igem.org

(Difference between revisions)
 
(48 intermediate revisions not shown)
Line 75: Line 75:
<script type="text/javascript"        src ="https://2012.igem.org/common/jquery-latest.min.js"></script>
<script type="text/javascript"        src ="https://2012.igem.org/common/jquery-latest.min.js"></script>
<script type="text/javascript"        src ="https://2012.igem.org/common/tablesorter/jquery.tablesorter.min.js"></script>
<script type="text/javascript"        src ="https://2012.igem.org/common/tablesorter/jquery.tablesorter.min.js"></script>
 +
<script src="http://webglmol.sourceforge.jp/glmol/js/Three49custom.js"></script>
 +
<script type="text/javascript" src="http://webglmol.sourceforge.jp/glmol/js/GLmol.js"></script>
         <link rel="stylesheet" type="text/css" href="https://2012.igem.org/common/tablesorter/themes/groupparts/style.css" />
         <link rel="stylesheet" type="text/css" href="https://2012.igem.org/common/tablesorter/themes/groupparts/style.css" />
Line 81: Line 83:
<script type="text/javascript"        src ="https://2012.igem.org/forum/forum_scripts.js"></script>
<script type="text/javascript"        src ="https://2012.igem.org/forum/forum_scripts.js"></script>
 +
<!-- slide script starts here -->
 +
<script type="text/javascript">
 +
function slider() {
 +
    var $current = $('#slides IMG.current');
 +
 +
    if ( $current.length == 0 ) $current = $('#slides IMG:last');
 +
    var $next =  $current.next().length ? $current.next()
 +
        : $('#slides IMG:first');
 +
 +
      $current.addClass('previous');
 +
    $next.css({opacity: 0.0})
 +
        .addClass('current')
 +
        .animate({opacity: 1.0}, 2500, function() {
 +
            $current.removeClass('current previous');
 +
        });
 +
 +
var $navicurrent = $('.progress li.current');
 +
if($navicurrent.length ==0) $navicurrent = $('.progress li:last');
 +
var $navinext = $navicurrent.next().length ? $navicurrent.next() : $('.progress li:first');
 +
$navicurrent.addClass('previous');
 +
$navicurrent.removeClass('current previous');
 +
$navinext.addClass('current').animate({opacity: 1.0}, 2500, function() {
 +
        });
 +
 +
$('.progress li').click(function(){
 +
   
 +
var $ncurrent = $(this);
 +
        $('.progress li').removeClass('current').removeClass('previous');
 +
        $('#slides IMG').removeClass('current').removeClass('previous');
 +
        $ncurrent.addClass('current');
 +
        var ncurrentindex = $ncurrent.index();
 +
if(  ncurrentindex=== 0) $current = $('#slides IMG:first');
 +
        if(  ncurrentindex=== 1) $current = $('#slides IMG:eq(1)');
 +
        if(  ncurrentindex=== 2) $current = $('#slides IMG:eq(2)');
 +
        if(  ncurrentindex=== 3) $current = $('#slides IMG:last');
 +
        $current.addClass('current');
 +
 +
});
 +
 +
}
 +
$(function() {
 +
    setInterval( "slider()", 5000 );
 +
});
 +
 +
$(document).ready(function(){
 +
 +
  $('a.lightbox').click(function(e) {
 +
    $('body').css('display', 'inherit'); // hide scrollbars!
 +
 +
    $('<div id="overlay"></div>')
 +
    .css('opacity', '0')
 +
      .animate({'opacity': '0.7'}, 'slow')
 +
    .click(function(){
 +
      removeLightbox();
 +
      })
 +
      .appendTo('body');
 +
 +
    $('<div id="lightbox"></div>')
 +
      .hide()
 +
      .appendTo('body');
 +
 +
    $('<img>')
 +
      .attr('src', $(this).attr('href'))
 +
      .load(function() {
 +
        positionLightboxImage();
 +
      })
 +
      .click(function() {
 +
        removeLightbox();
 +
      })
 +
      .appendTo('#lightbox');
 +
 +
    return false;
 +
  });
 +
});
 +
 +
function positionLightboxImage() {
 +
  var top = ($(window).height()  - $('#lightbox').height())/2;
 +
  var left = ($(window).width() - $('#lightbox').width())/2;
 +
 +
  $('#lightbox')
 +
    .css({
 +
      'top': top,
 +
      'left':left-80
 +
    })
 +
    .fadeIn();
 +
}
 +
 +
function removeLightbox() {
 +
 +
  $('#overlay, #lightbox')
 +
    .fadeOut('slow', function() {
 +
      $(this).remove();
 +
      $('body').css('overflow-y', 'auto'); // show scrollbars!
 +
    });
 +
}
 +
 +
 +
</script>
 +
<!-- slide script ends here -->
<style type="text/css">
<style type="text/css">
<!-- beginning of slider style -->
<!-- beginning of slider style -->
Line 89: Line 190:
min-height:100%; /* real browsers */
min-height:100%; /* real browsers */
}
}
-
#content { z-index: 1; background-color: transparent; border: none; padding: 0; margin: 0; width: 100%; overflow: hidden; margin-top: -45px !important;
+
#content { z-index: 1; background-color: transparent; border: none; padding: 0; margin: 0; width: 100%; overflow: hidden; top:-3px; margin-top: -45px !important;
height:auto !important; /* real browsers */
height:auto !important; /* real browsers */
height:100%; /* IE6: treaded as min-height*/
height:100%; /* IE6: treaded as min-height*/
Line 106: Line 207:
#menubar.right-menu { width:300px; display:block; float:left; margin-top:-80px; border: none;}
#menubar.right-menu { width:300px; display:block; float:left; margin-top:-80px; border: none;}
.right-menu ul { border: none; width: 300px;}
.right-menu ul { border: none; width: 300px;}
-
#footer-box { background-color: #216085; border: none; width: 100%; height: 100%; margin: -10px auto 0 auto; padding: 20px 0;}
+
#footer-box { background-color: #216085; border: none; width: 850px; height: 30px; margin: -10px auto 0 auto; padding: 20px 0;}
.visualClear { display: none; }
.visualClear { display: none; }
#footer { border: none; width: 850px; margin: 0 auto; padding: 0;}
#footer { border: none; width: 850px; margin: 0 auto; padding: 0;}
Line 118: Line 219:
h3#siteSub { display: none;}
h3#siteSub { display: none;}
#contentSub {display: none;}
#contentSub {display: none;}
-
p:first-child { display: none;}
+
p:first-child { display: block;}
h1{border:none; width: 100%; clear: both;}
h1{border:none; width: 100%; clear: both;}
-
<!-- end of slides style -->
 
-
 
-
body {
 
-
    background-color: #ffffff;
 
-
    color: black;
 
-
    font: x-small sans-serif;
 
-
    margin: 0;
 
-
    padding: 0;
 
-
    height:100%;
 
-
#    height:auto !important;
 
-
}
 
#globalWrapper {
#globalWrapper {
     position: relative;
     position: relative;
     font-size: 127%;
     font-size: 127%;
-
     width: 100%;
+
     width: 850px //100%;
     margin: 0;
     margin: 0;
     padding: 0;
     padding: 0;
     padding-bottom: 10px;
     padding-bottom: 10px;
-
    background-color: #ffffff;
+
#    background:url(https://static.igem.org/mediawiki/2012/c/cc/UCD_Bg_1.jpg);
-
#   height:100%;
+
#    background-repeat: no-repeat;
-
 
+
#   background-attachment: fixed;
 +
#   background-size:100% auto;
}
}
Line 149: Line 240:
     margin: 0 auto;
     margin: 0 auto;
     padding: 5px 5px 5px 5px;;
     padding: 5px 5px 5px 5px;;
-
     background: white;
+
     background: transparent;
     color: black;
     color: black;
-
    border-left: 1px solid #ffffff;
+
#    border-left: 1px solid #ffffff;
-
    border-right: 1px solid #ffffff;
+
#    border-right: 1px solid #ffffff;
-
    line-height: 1.5em;
+
#    line-height: 1.5em;
-
#    z-index: 2;
+
}
}
 +
 +
 +
body {
 +
background-color: rgba(255,255,255,1);
 +
background-image: url('https://static.igem.org/mediawiki/2012/c/cc/UCD_Bg_1.jpg');
 +
background-size: 100%;
 +
background-repeat: no-repeat;
 +
background-attachment:fixed;
 +
 +
    color: black;
 +
    font: x-small sans-serif;
 +
    margin: 0;
 +
    padding: 0;
 +
#    height:100%;
 +
}
 +
#topmenubar {
#topmenubar {
Line 211: Line 317:
         position:relative;
         position:relative;
         height:42px;
         height:42px;
-
         width: 856px;
+
         width: 850px;
         margin-left: -2px;
         margin-left: -2px;
         display:block;
         display:block;
Line 220: Line 326:
         overflow:hidden;
         overflow:hidden;
         padding:0px;  
         padding:0px;  
-
         margin:0px;  
+
         margin:2px solid transparent;  
         list-style-type:none;  
         list-style-type:none;  
         height:24px;
         height:24px;
 +
        width: 107px;
         list-style-image:none;
         list-style-image:none;
-
         background:#004b85;
+
         background: #004b85;
}
}
#newnavi .newmenu li    {
#newnavi .newmenu li    {
         float:right;  
         float:right;  
-
        height:24px;
+
         background: #004b85;
-
        width: 103px;
+
         text-align:center;
-
         background:#004b85;
+
-
         border-left: solid 2px white;
+
-
        border-right: solid 2px white;
+
}
}
#newnavi .newmenu .selected {
#newnavi .newmenu .selected {
Line 238: Line 342:
}
}
#newnavi .newmenu li a  {
#newnavi .newmenu li a  {
-
text-align:center; vertical-align:middle; width:82px; padding:5px 10px 5px 10px; display:block; color:#fff; text-decoration:none; font:14px arial, verdana, sans-serif; font-weight: bold;
+
text-align:center; vertical-align:middle; width:86px; padding:5px 10px 5px 10px; display:block; color:#fff; text-decoration:none; font:14px arial, verdana, sans-serif; font-weight: bold;
}
}
#newnavi .newmenu li:hover a {
#newnavi .newmenu li:hover a {
Line 244: Line 348:
}
}
#newnavi .newmenu ul    {
#newnavi .newmenu ul    {
-
position:absolute; left:-9999px; top:-9999px; list-style-type:none; list-style-image:none; margin:0px; border:0px; padding:0px; background:lavender;
+
position:absolute; left:-9999px; top:-9999px; list-style-type:none; list-style-image:none; margin:0px; border:0px; padding:0px; background: #05bcea;
}
}
#newnavi .newmenu li:hover      {
#newnavi .newmenu li:hover      {
Line 250: Line 354:
}
}
#newnavi .newmenu li:hover ul  {
#newnavi .newmenu li:hover ul  {
-
left:0px; top:23px; background:lavender;  width:103px; opacity:0.7; z-index:3;
+
left:0px; top:26px; background: #05bcea;  width:150px; opacity:1.0 ; z-index:4;
}
}
#newnavi .newmenu li:hover ul li        {
#newnavi .newmenu li:hover ul li        {
-
         height:17px;
+
          
-
        border:none;
+
         background: #05bcea;
-
         background:lavender;
+
}
}
#newnavi .newmenu li:hover ul li a      {
#newnavi .newmenu li:hover ul li a      {
-
text-align:center; height:17px; padding:0px;display:block; font-size:12px; width:103px; line-height:16px; text-indent:0px; color:#444;  
+
text-align:left; padding:5px 0px 5px 0px;display:block; font-size:12px; width:150px; text-indent:10px; color:#444;  
-
background-color:transparent; text-decoration:none; border:0px solid transparent;       
+
background-color: #05bcea; text-decoration:none;       
}
}
#newnavi .newmenu li:hover ul li a:hover {
#newnavi .newmenu li:hover ul li a:hover {
-
height:18px; background:silver; color:#000; border:solid 0px #444;    
+
background: #05bcea; color:#ffffff; width:150px;     
 +
}
 +
 
 +
 
 +
#newnavi .newmenu li ul li ul {
 +
        height:40px;
 +
        left:299px;
 +
        top:-26px;
 +
        margin: 0;
 +
padding: 0;
 +
list-style: none;
 +
width: 150px;
 +
        position:absolute;
 +
display:none;
 +
}
 +
 
 +
#newnavi .newmenu li ul li ul li {
 +
position:relative;
 +
background: #05bcea;
 +
width: 150px;
 +
}
 +
 
 +
#newnavi .newmenu li ul li ul li a {
 +
 
 +
padding:5px 0px 5px 0px;display:block; font-size:12px; width:100px; text-indent:10px; color:#444;
 +
background-color: #05bcea; text-decoration:none;
 +
}
 +
 
 +
#newnavi .newmenu li ul li:hover ul {
 +
display:block;
 +
color:#ffffff;
 +
width:150px;
 +
background:transparent;
 +
}
 +
 
 +
#newnavi .newmenu li ul li:hover ul li {
 +
top:-25px;
 +
left:150px;
 +
margin-bottom:-1px;
 +
height:26px;
 +
}
 +
 
 +
#newnavi .newmenu li ul li:hover ul li a {
 +
 
 +
padding:5px 0px 5px 0px;display:block; font-size:12px; text-indent:10px; color:#444;
 +
background-color: #05bcea; text-decoration:none;border:0px solid;
 +
}
 +
 
 +
 
 +
#slides {
 +
    position:relative;
 +
    height:280px;
 +
    width: 840px
 +
   
 +
}
 +
 
 +
#slides IMG {
 +
        position:absolute;
 +
        opacity:0.0;
 +
        top:0px;   
 +
        left:0px;
 +
        z-index:1;   
 +
}
 +
 
 +
#slides .current {
 +
    z-index:3;
 +
    opacity:1.0;
 +
}
 +
 
 +
#slides .previous {
 +
    z-index:2;
 +
}
 +
 
 +
.progress {
 +
 
 +
margin-top:-270px;
 +
margin-left:20px;
 +
position:absolute;
 +
 
 +
}
 +
 
 +
.progress li{
 +
border:1px solid rgba(0,0,0,0.9);
 +
border-radius:3px;
 +
margin-left: 5px;
 +
float:left;
 +
display:block;
 +
position:relative;
 +
z-index:10;
 +
background-color:rgba(255,255,255,0.4);
 +
}
 +
 
 +
.progress li a{
 +
    padding-left: 6px;
 +
    padding-right: 6px;
 +
}
 +
 
 +
.progress li a:hover {
 +
text-decoration:none;
 +
}
 +
 
 +
.progress .current {
 +
background-color:white;
 +
}
 +
 
 +
#myleftrightbox
 +
{
 +
position:relative;
 +
width:850px;
 +
float:left;
 +
background-color:transparent;
 +
margin-top: 0px;
 +
margin-left:3.75px solid transparent;
 +
margin-right:3.75px solid transparent;
 +
border-radius: 4px;
 +
padding: 0px;
 +
font: sans-serif;
 +
font-size: 13px;
 +
color: white;
 +
}
 +
 
 +
#myleftrightbox .fourboxes
 +
{
 +
width:191px;
 +
float:left;
 +
background-color:rgba(143,143,143,0.7);
 +
margin-top: 15px;
 +
margin-left:3.75px solid #e8eff1;
 +
margin-right:3.75px solid #e8eff1;
 +
margin-bottom:0px;
 +
border-radius: 4px;
 +
padding: 8.5px;
 +
font: sans-serif;
 +
font-size: 13px;
 +
color: white;
 +
}
 +
 
 +
#myleftrightbox .fourboxes-1
 +
{
 +
width:191px;
 +
float:left;
 +
background-color:rgba(143,143,143,0.7);
 +
margin-top: 15px;
 +
margin-left:3.75px solid #e8eff1;
 +
margin-right:3.75px solid #e8eff1;
 +
margin-bottom:0px;
 +
border-radius: 4px;
 +
padding: 8.5px;
 +
font: sans-serif;
 +
font-size: 13px;
 +
color: white;
 +
}
 +
 
 +
#myleftrightbox .fourboxes-2
 +
{
 +
width:191px;
 +
float:left;
 +
background-color:rgba(143,143,143,0.7);
 +
margin-top: 15px;
 +
margin-left:3.75px solid #e8eff1;
 +
margin-right:3.75px solid #e8eff1;
 +
margin-bottom:0px;
 +
border-radius: 4px;
 +
padding: 8.5px;
 +
font: sans-serif;
 +
font-size: 13px;
 +
color: white;
 +
}
 +
 
 +
#myleftrightbox .fourboxes-3
 +
{
 +
width:191px;
 +
float:left;
 +
background-color:rgba(143,143,143,0.7);
 +
margin-top: 15px;
 +
margin-left:3.75px solid #e8eff1;
 +
margin-right:3.75px solid #e8eff1;
 +
margin-bottom:0px;
 +
border-radius: 4px;
 +
padding: 8.5px;
 +
font: sans-serif;
 +
font-size: 13px;
 +
color: white;
 +
}
 +
 
 +
#myleftrightbox .fourboxes-4
 +
{
 +
width:191px;
 +
float:left;
 +
background-color:rgba(143,143,143,0.7);
 +
margin-top: 15px;
 +
margin-left:3.75px solid #e8eff1;
 +
margin-right:3.75px solid #e8eff1;
 +
margin-bottom:0px;
 +
border-radius: 4px;
 +
padding: 8.5px;
 +
font: sans-serif;
 +
font-size: 13px;
 +
color: white;
 +
}
 +
 
 +
#myleftrightbox .fourboxes-1:hover {
 +
background-color:green;
 +
}
 +
#myleftrightbox .fourboxes-2:hover {
 +
background-color:orange;
 +
}
 +
#myleftrightbox .fourboxes-3:hover {
 +
background-color:#105ec5;
 +
}
 +
#myleftrightbox .fourboxes-4:hover {
 +
background-color:#4a4a4a;
 +
}
 +
 
 +
 
 +
#myleftrightbox .fourboxes2
 +
{
 +
width:191px;
 +
float:left;
 +
background-color:rgba(143,143,143,0.7);
 +
margin-top: 5px;
 +
margin-left:3.75px solid #e8eff1;
 +
margin-right:3.75px solid #e8eff1;
 +
margin-bottom:0px;
 +
border-radius: 4px;
 +
padding: 8.5px;
 +
font: sans-serif;
 +
font-size: 13px;
 +
color: white;
 +
}
 +
 
 +
#myleftrightbox .spacebox
 +
{
 +
float:left;
 +
width:5px;
 +
 
 +
}
 +
#myleftbox
 +
{
 +
position:relative;
 +
width:625px;
 +
float:left;
 +
background-color:;
 +
margin-top: 0px;
 +
border-radius: 4px;
 +
padding: 0px;
 +
font: sans-serif;
 +
font-size: 13px;
 +
color: white;
 +
}
 +
#myleftbox .smallbox
 +
{
 +
width:604px;
 +
float:left;
 +
background-color:#bfbfbf;
 +
margin-top: 15px;
 +
border-radius: 4px;
 +
padding: 15px 15px 15px 15px;
 +
font: sans-serif;
 +
font-size: 13px;
 +
line-height:1.5em;
 +
color:black;
 +
}
 +
#myleftbox .smallbox h1
 +
{
 +
color:#004b85;
 +
font-weight:bold;
 +
}
 +
#myleftbox .smallbox p
 +
{
 +
color: #004b85;
 +
font-weight:bold;
 +
}
 +
#myleftbox .smallboxsite
 +
{
 +
width:604px;
 +
float:left;
 +
background-color:rgba(191,191,191,0.6 );
 +
margin-top: 17px;
 +
border-radius: 4px;
 +
padding: 15px 15px 15px 15px;
 +
font: sans-serif;
 +
font-size: 13px;
 +
line-height:1.5em;
 +
color:black;
 +
 
 +
}
 +
#myleftbox .threeboxes
 +
{
 +
width:191px;
 +
float:left;
 +
background-color:rgba(143,143,143,0.7);
 +
margin-top: 15px;
 +
border-radius: 4px;
 +
border-left:2px solid #e8eff1;
 +
border-right:2px solid #e8eff1;
 +
padding: 7px 7px 7px 7px;
 +
font: sans-serif;
 +
font-size: 13px;
 +
color: white;
 +
}
 +
 
 +
#myleftbox .threeboxes2
 +
{
 +
width:191px;
 +
float:left;
 +
background-color:rgba(143,143,143,0.7);
 +
margin-top: 5px;
 +
border-radius: 4px;
 +
border-left:2px solid #e8eff1;
 +
border-right:2px solid #e8eff1;
 +
padding: 7px 7px 7px 7px;
 +
font: sans-serif;
 +
font-size: 13px;
 +
color: white;
 +
}
 +
 
 +
#myleftbox .threeleft
 +
{
 +
width:175px;
 +
float:left;
 +
background-color:#ba9108;
 +
margin-top: -52px;
 +
border-radius: 4px;
 +
padding: 15px 15px 15px 15px;
 +
font: sans-serif;
 +
font-size: 13px;
 +
color: white;
 +
}
 +
 
 +
#myleftbox .threecenter
 +
{
 +
width:175px;
 +
#float:left;
 +
background-color:#ba9108;
 +
margin-top: -52px;
 +
border-radius: 4px;
 +
padding: 15px 15px 15px 15px;
 +
font: sans-serif;
 +
font-size: 13px;
 +
color: white;
 +
}
 +
#myleftbox .threeright
 +
{
 +
width:175px;
 +
float:right;
 +
background-color:#ba9108;
 +
margin-top: -52px;
 +
border-radius: 4px;
 +
padding: 15px 15px 15px 15px;
 +
font: sans-serif;
 +
font-size: 13px;
 +
color: white;
 +
}
 +
 
 +
 
 +
 
 +
#myleftbox .twoboxes
 +
{
 +
width:295px;
 +
height:120px;
 +
float:left;
 +
background-color:rgba(143,143,143,0.7);
 +
margin-top: 15px;
 +
border-radius: 4px;
 +
border-left:2px solid #e8eff1;
 +
border-right:2px solid #e8eff1;
 +
padding: 7px 7px 7px 7px;
 +
font: sans-serif;
 +
font-size: 13px;
 +
color: white;
 +
}
 +
 
 +
#myleftbox .twoboxes1
 +
{
 +
width:295px;
 +
height:120px;
 +
float:left;
 +
background-color:rgba(143,143,143,0.7);
 +
margin-top: 15px;
 +
border-radius: 4px;
 +
border-left:1px solid #e8eff1;
 +
border-right:2px solid #e8eff1;
 +
padding: 7px 7px 7px 7px;
 +
font: sans-serif;
 +
font-size: 13px;
 +
color: white;
 +
left:9px;
 +
}
 +
 
 +
#myleftbox .twoboxes:hover {
 +
background-color:red;
 +
}
 +
 
 +
#myleftbox .twoboxes1:hover {
 +
background-color:green;
 +
}
 +
 
 +
#sponsorbox
 +
{
 +
position: relative;
 +
width: 208px;
 +
margin-top:15px;
 +
float: right;
 +
height: auto;
 +
}
 +
 
 +
#sponsorbox .sponsorfloat
 +
{
 +
left:-3px;
 +
width:198px;
 +
float: right;
 +
background-color: #004b85;
 +
margin-top: 0px;
 +
margin-bottom:5px;
 +
border-radius: 4px;
 +
padding: 5px 5px 5px 5px;
 +
font: sans-serif;
 +
font-size: 13px;
 +
color: rgba(225,225,225,1);
 +
}
 +
 
 +
#sponsorbox .sponsorfloat h2
 +
{
 +
#color:#ba9108;
 +
color:white;
 +
font-size:130%;
 +
}
 +
 
 +
#sponsorbox .sponsorfloat a
 +
{
 +
color:white;
 +
font: sans-serif;
 +
}
 +
 
 +
#sponsorbox .sponsorfloat p
 +
{
 +
font: sans-serif;
 +
}
 +
.newsAnnouncement
 +
{
 +
width:205px;
 +
height:110px;
 +
overflow-x:hidden;
 +
overflow-y:scroll;
}
}
Line 283: Line 830:
font-weight:bold;
font-weight:bold;
font-size: 40px;
font-size: 40px;
-
color: #004b85;;
+
color: #016D8B;;
-
left:10px;
+
-
}
+
-
 
+
-
.floatbox3 hp{
+
-
font-weight:bold;
+
-
font-size: 20px;
+
-
color: #004b85;;
+
left:10px;
left:10px;
}
}
Line 318: Line 858:
     padding: 0 .0em;}
     padding: 0 .0em;}
-
 
+
#catlinks {
 +
background-color:transparent;
 +
border:1px solid transparent;
 +
}
 +
#footpage {
 +
background-color:transparent;
 +
}
 +
#footer-box {
 +
background-color:rgba(191,191,191,0.5);
 +
margin-top:0px;
 +
}
#footer-box-1 {
#footer-box-1 {
          
          
Line 327: Line 877:
         border: 1px solid #444444;
         border: 1px solid #444444;
}
}
 +
#tweets {
 +
    width: 203px;
 +
    margin: 0 auto;
 +
    font: sans-serif;
 +
    font-size: 12px;
 +
}
 +
 +
#tweets .twtr-widget,
 +
#tweets .twtr-doc {
 +
 +
    width: 100%;
 +
    height: auto;
 +
}
 +
 +
#tweets .twtr-hd {
 +
    display:none;
 +
    background: #d40;
 +
    color: #fff;
 +
    -moz-border-radius: 4px 4px 0 0;
 +
    border-radius: 4px 4px 0 0;
 +
    font-family: sans-serif !important;
 +
    }
 +
 +
#tweets .twtr-hd *,
 +
#tweets .twtr-hd h4 a {
 +
 +
    background: #d40 !important;
 +
    font: sans-serif !important;
 +
}
 +
 +
#tweets .twtr-hd h3,
 +
#tweets .twtr-hd h4 {
 +
 +
    font-weight: normal;
 +
    text-align: left;
 +
    margin:0;
 +
}
 +
 +
#tweets .twtr-hd h3 {
 +
       
 +
    background: #fff !important;
 +
    color: #333 !important;
 +
    font: sans-serif;
 +
    font-size: 1.0em !important;
 +
    padding: 0.2em !important;
 +
    -moz-border-radius: 0px;
 +
    border-radius: 0px;
 +
 +
}
 +
 +
#tweets .twtr-tweet {
 +
 +
    background: #fff;
 +
    font: sans-serif !important;
 +
    font-size: 12px;
 +
}
 +
 +
#tweets .twtr-tweet a:link,
 +
#tweets .twtr-tweet a:visited,
 +
#tweets .twtr-tweet a:hover {
 +
 +
    color: #c40 !important;
 +
 +
}
 +
 +
#tweets .twtr-tweet .twtr-tweet-text {
 +
 +
    border-bottom: 1px dashed #666 !important;
 +
    padding-bottom: 4px !important;
 +
    font: sans-serif;
 +
    font-size:12px;
 +
}
 +
 +
#tweets .twtr-tweet .twtr-tweet-text p {
 +
 +
    color: #333 !important;
 +
    font: sans-serif;
 +
    font-size: 12px;
 +
 +
}
 +
 +
#overlay {
 +
  z-index:2;
 +
  position:fixed;
 +
  top: 0;
 +
  left: 0;
 +
  height: 100%;
 +
  width: 100%;
 +
  background: black no-repeat scroll center center;
 +
}
 +
 +
#lightbox {
 +
  position: fixed;
 +
  width:840;
 +
  z-index:2;
 +
}
 +
 +
table {
 +
background-color: #bfbfbf;
 +
}
 +
 +
td {
 +
padding: 8px;
 +
}
 +
 +
captiontext {
 +
font-size: 11px;
 +
}
</style>
</style>
Line 342: Line 1,000:
    <ul>
    <ul>
  <li style='color:#014457; cursor:default'><a>teams</a></li>
  <li style='color:#014457; cursor:default'><a>teams</a></li>
-
                    <li class='selected'        ><a href="https://2012.igem.org/Team:UC_Davis">Page</a></li>
+
                    <li class='selected'        ><a href="https://2012.igem.org/Team:UC_Davis/Project/Protein_Engineering">Page</a></li>
-
 
+
                 <li class='new'><a href="https://2012.igem.org/wiki/index.php?title=Talk:Team:UC_Davis/Project/Protein_Engineering&amp;action=edit&amp;redlink=1">Discussion</a></li>
-
                 <li class='new'><a href="https://2012.igem.org/wiki/index.php?title=Talk:Team:UC_Davis&amp;action=edit&amp;redlink=1">Discussion</a></li>
+
               <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis/Project/Protein_Engineering&action=edit">Edit</a></li>
               <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis/Project/Protein_Engineering&action=edit">Edit</a></li>
-
                           <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis&amp;action=history">History</a></li>
+
                           <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis/Project/Protein_Engineering&amp;action=history">History</a></li>
-
                 <li><a href="https://2012.igem.org/Special:MovePage/Team:UC_Davis">Move</a></li>
+
                 <li><a href="https://2012.igem.org/Special:MovePage/Team:UC_Davis/Project/Protein_Engineering">Move</a></li>
-
                           <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis&amp;action=watch">Watch</a></li>
+
                           <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis/Project/Protein_Engineering&amp;action=watch">Watch</a></li>
  <li><a href="https://igem.org/Login">Log in</a></li>
  <li><a href="https://igem.org/Login">Log in</a></li>
Line 355: Line 1,012:
   <div id="newnavi">
   <div id="newnavi">
     <ul class="newmenu">
     <ul class="newmenu">
-
        <li ><a href="https://2012.igem.org/" title="Back to iGEM">iGEM</a></li>
+
    <li ><a target="new" href="https://2012.igem.org/" title="Back to iGEM">iGEM</a>
 +
          <ul>
 +
          <li><a target="new" href="https://2012.igem.org/">Main iGEM</a></li>
 +
          <li><a href="https://2012.igem.org/Team:UC_Davis/Criteria">Criteria</a></li>
 +
          <li><a href="https://2012.igem.org/Team:UC_Davis/Human_Practices">Human Practices</a></li>
 +
          </ul>
 +
        </li>
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Attributions" title="Attributions">Attributions</a></li>
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Attributions" title="Attributions">Attributions</a></li>
-
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Data" title="Data">Data</a>
+
         <li ><a title="https://2012.igem.org/Team:UC_Davis/Data" title="Data">Data</a>
           <ul>
           <ul>
-
             <li ><a href="./Data.htm ">Data 1</a></li>
+
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Cutinase_Activity" title="Data">Cutinase Activity</a></li>
-
             <li ><a href="./Data.htm ">Data 2</a></li>
+
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol"
-
            <li ><a href="./Data.htm ">Data 3</a></li>
+
title="Data">Ethylene Glycol</a></li>
-
        </ul>
+
<li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Modeling"
 +
title="Data">Modeling</a></li>
 +
 
 +
            <li ><a href="https://2012.igem.org/Team:UC_Davis/Parts">Parts</a></li>
 +
          </ul>
         </li>
         </li>
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook" title="Notebook">Notebook</a>
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook" title="Notebook">Notebook</a>
           <ul>
           <ul>
-
             <li ><a href="./Notebook.htm ">Week 1</a></li>
+
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook">Notebook</a></li>
-
             <li ><a href="./Notebook.htm ">Week 2</a></li>
+
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook/Protocols ">Protocols</a></li>
-
             <li ><a href="./Notebook.htm ">Week 3</a></li>
+
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook/Gallery">Gallery</a></li>
           </ul>
           </ul>
         </li>
         </li>
Line 378: Line 1,045:
         <li class="selected"><a href="https://2012.igem.org/Team:UC_Davis/Project" title="Project">Project</a>
         <li class="selected"><a href="https://2012.igem.org/Team:UC_Davis/Project" title="Project">Project</a>
           <ul>
           <ul>
-
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Project/Strain">Strain</a></li>
+
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Project">Project Overview</a></li>
-
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Project/Catalyst">Catalyst</a></li>
+
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Project/Catalyst">Module Engineering</a></li>
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Project/Protein_Engineering">Protein Engineering</a></li>
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Project/Protein_Engineering">Protein Engineering</a></li>
 +
            <li ><a title="https://2012.igem.org/Team:UC_Davis/Project/Strain">Chassis Engineering </a>
 +
  <ul>
 +
                <li><a href="https://2012.igem.org/Team:UC_Davis/Project/Strain">Background</a></li>
 +
        <li><a href="https://2012.igem.org/Team:UC_Davis/Project/Directed_Evolution">Directed Evolution</a></li>
 +
                <li><a href="https://2012.igem.org/Team:UC_Davis/Project/Our_Strain">Rational Engineering </a></li>
 +
</ul>
 +
</li>
           </ul>
           </ul>
         </li>
         </li>
Line 387: Line 1,061:
     </ul>
     </ul>
   </div>
   </div>
 +
<!-- slide show starts here
 +
<div id="slides">
 +
 +
    <img  src="https://static.igem.org/mediawiki/2012/c/cf/UCD_slide1.jpg" width="850" height="280" alt="" class="current" />
 +
    <img  src="https://static.igem.org/mediawiki/2012/e/e3/UCD_Slide_2.jpg" width="850" height="280" alt="" />
 +
    <img  src="https://static.igem.org/mediawiki/2012/d/d9/UCD_Slide_3.jpg" width="850" height="280" alt="" />
 +
    <img  src="https://static.igem.org/mediawiki/2012/a/a4/UCD_Slide_4.jpg" width="850" height="280" alt="" />
 +
</div>
 +
    <ul class="progress">
 +
<li class="current"><a href="#n_0">1</a> </li>
 +
<li><a href="#n_1">2</a> </li>
 +
        <li><a href="#n_2">3</a> </li>
 +
        <li><a href="#n_3">4</a> </li>
 +
    </ul>
 +
slide show ends here -->
 +
 +
<img src="https://static.igem.org/mediawiki/2012/1/17/UCD_protein_engineering_banner.jpg">
 +
 +
<div id="bodyContent"> 
 +
<!--  <div id="contentSub"></div> -->
 +
<!--  </div> -->
 +
<!-- float: right; first try -->
 +
<!-- 1st row four boxes starts here -->
 +
<div id="myleftrightbox">
 +
<div id="myleftrightbox"  class="fourboxes-1">
 +
<a href="https://2012.igem.org/Team:UC_Davis/Project"><img src="https://static.igem.org/mediawiki/2012/7/73/UCD_project_small_banner.jpg
 +
"></a>
 +
</div>
 +
<div id="myleftrightbox"  class="spacebox">
 +
<p></p>
 +
</div>
 +
<div id="myleftrightbox"  class="fourboxes-2">
 +
<a href="https://2012.igem.org/Team:UC_Davis/Project/Catalyst"><img src="https://static.igem.org/mediawiki/2012/5/55/UCD_modular_small_banner.jpg
 +
"></a>
 +
</div>
 +
<div id="myleftrightbox"  class="spacebox">
 +
<p></p>
 +
</div>
 +
<div id="myleftrightbox"  class="fourboxes-3">
 +
<a href="https://2012.igem.org/Team:UC_Davis/Project/Strain"><img src="https://static.igem.org/mediawiki/2012/e/ec/UCD_chassis_small_banner.jpg
 +
"></a>
 +
</div>
 +
<div id="myleftrightbox"  class="spacebox">
 +
<p></p>
 +
</div>
 +
<div id="myleftrightbox"  class="fourboxes-4">
 +
<a href="https://2012.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2012/1/18/UCD_criteria_small_banner.jpg"></a>
 +
</div>
 +
</div>
 +
<!-- 1st row four boxes ends here -->
 +
 +
 +
<!-- <div id="sponsorbox" style="width:220px; float:right;"> -->
 +
 +
<div id="sponsorbox">
 +
<div id="sponsorbox" class="sponsorfloat">
 +
<!-- twitter starts here -->
 +
</script>
 +
<div id="tweets">
 +
<center>
 +
<h2>UCDavis iGEM Tweets</h2>
 +
</center>
 +
<script charset="utf-8" src="http://widgets.twimg.com/j/2/widget.js"></script>
 +
<script>
 +
new TWTR.Widget({
 +
  version: 2,
 +
  type: 'profile',
 +
  rpp: 10,
 +
  interval: 30000,
 +
  width: 203,
 +
  height: 100,
 +
 
 +
  theme: {
 +
    shell: {
 +
      background: '#004b85',
 +
      color: '#ffffff'
 +
    },
 +
    tweets: {
 +
      background: '#004b85',
 +
      color: '#ffffff',
 +
      links: '#4aed05'
 +
    }
   
   
-
+
  },
-
<img src="http://img.photobucket.com/albums/v26/bluemelon/protein_banner1.jpg" width="850" height="228">
+
  features: {
 +
    scrollbar: true,
 +
    loop: false,
 +
    live: true,
 +
    behavior: 'all'
 +
  }
 +
}).render().setUser('UCDavisiGEM').start();
-
  <div id="bodyContent">  
+
</script>
-
            <div id="contentSub"></div>
+
</div>
 +
<!-- twitter ends here -->
 +
</div>
-
<br>
+
<div id="sponsorbox" class="sponsorfloat">
 +
<center>
 +
<h2>Our Sponsors</h2>
 +
<a href="http://www.novozymes.com/en/Pages/default.aspx" target="_blank"><img src="https://static.igem.org/mediawiki/2011/2/21/UCD_Novozymes-logo.jpg" width="200"></a>
 +
</center>
-
  <div class="floatbox3">
+
<center>
-
<h1> Protein Engineering </h1>
+
<a href="http://engineering.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2011/f/f6/UCD_CoE.png" width="200" height="40"></a>
-
<article>
+
</center>
-
text
+
<center>
 +
<a href="http://biosci.ucdavis.edu/index_js.html" target="_blank"><img src="https://static.igem.org/mediawiki/2011/b/b1/UCD_biosci_sponsor.jpg" width="200" height="90"></a>
 +
</center>
-
</article>
+
<center>
-
  </div>
+
<a href="http://www.genomecenter.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2011/1/1b/UCD_Genome_center_sponsor.jpg" width="200" height="60"></a>
 +
</center>
-
<br>
+
<center>
-
<div class="floatbox3">
+
<a href="http://www.cs.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/6/6b/UCD_Computer_sponsor.jpg" width="200"></a>
-
<hp>Background</hp>
+
</center>
 +
 
 +
<center>
 +
<a href="http://www.bme.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2011/4/40/UCD_BME_logo_minimal_copy.png" width="200 height="70"></a>
 +
</center>
 +
 
 +
<center>
 +
<a href="http://www.fishersci.com" target="_blank"><img src="https://static.igem.org/mediawiki/2011/a/a4/UCD_Fisher_Logo.gif" width="200"></a>
 +
</center>
 +
 
 +
<center>
 +
<a href="http://www.arcadiabio.com/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/4/46/UCD_Arcadia_sponsor.jpg
 +
" width="200"></a>
 +
</center>
 +
 
 +
<center>
 +
<a href="http://provost.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/8/82/UCD_Provost_sponsor.jpg
 +
" width="200"></a>
 +
</center>
 +
 
 +
<center>
 +
<a href="http://www.research.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/9/99/UCD_Research_sponsor.jpg" width="200"></a>
 +
</center>
 +
 
 +
<center>
 +
<a href="http://ucomm.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/b/b4/UCD_Communications_sponsor.jpg" width="200"></a>
 +
</center>
 +
 
 +
<center>
 +
<a title="" target="_blank"><img src="https://static.igem.org/mediawiki/2012/2/25/UCD_Schultz_sponsor.jpg
 +
" width="200"></a>
 +
</center>
 +
 
 +
</div>
 +
</div>
 +
<!--  <br> -->
 +
<!-- div id="myleftbox" style="width:625px; float:left;" -->
 +
<div id="myleftbox">
 +
 
 +
<div class="smallbox" id="myleftbox">
 +
<h1>Protein Engineering</h1>
<article>
<article>
-
Beyond the general circuit outline for our degradation of PET, we also have two different protein engineering projects we performed. In the first project we constructed a more effective and higher yield form of the LC-Cutinase protein. In a second less related project we mutated the lac-repressor in order to change its ligand specificity. We changed its specificity to bind a harmful chemical called diuron instead of the natural IPTG in order to create a unique biosensor system.  
+
 
 +
Beyond the general circuit outline for our degradation of PET, there are also a protein engineering project we performed. In the project we constructed a more effective and higher yield form of the LC-Cutinase protein. We hoped to move toward a more realistic and economically feasible rate of degradation.  
</article></div>
</article></div>
 +
<br>
<br>
-
<div class="floatbox3">
+
<div class="smallbox" id="myleftbox">
-
<hp>What we're doing</hp>
+
<h1>LC-Cutinase Engineering</h1>
 +
<p>Confirming expectation of common results from previous mutations</p>
 +
 
<article>
<article>
-
<b>LC-Cutinase Engineering</b>
+
We had two goals with the engineering of cutinase: replicate mutations made in the paper by Silva et. al., which consisted of three residues mutated to alanines (1), as well as to generate our own chosen mutations. Before we decided to replicate Silva’s mutations we initially wanted to validate if we could expect the same results in our protein with the same mutations. In order to do this we wanted to compare best fit models (proteins with known structure who's sequence best matches an input sequence) generated for LC-Cutinase’s sequence and Silva’s Tfu_0883’s sequence. Replicating Silva et al. (1) we used <a target="new" href="http://swissmodel.expasy.org/">Swiss-Model</a> to generate a best fit model for LC-Cutinase, resulting in the protein 3visB with 61% homology. In Silva’s paper they received 1jfr, a model that was second best in LC-Cutinase’s output, with a 55% homology (1). This similarity between generated models and high homology rate gave support to the expectation of similar results from the same mutations.
 +
<br><br>
 +
<center><a href="https://static.igem.org/mediawiki/2012/c/cf/UCD_Figure_large-7.jpg" class="lightbox"><img src="https://static.igem.org/mediawiki/2012/1/15/UCD_Figure-7.png "> </a> </center><br><br>
<br>
<br>
-
We had two goals with the engineering of cutinase: replicate mutations made in the paper by Sulaiman et. al., which consisted of three residues mutated to alanines, as well as to generate our own chosen mutations. Before we decided to replicate Sulaiman’s mutations we initially wanted to validate if we could expect the same results in our protein with the same mutations. In order to do this we wanted to compare best fit models (proteins with known structure that best fit an input sequence) generated for LC-Cutinase’s sequence and Sulaiman’s Tfu_0883’s sequence. Replicating Sulaiman we used Swiss-Model to generate a best fit model for LC-Cutinase, resulting in the protein 3visB with 61% homology. In Sulaiman’s paper they received 1jfr, a model that was second best in LC-Cutinase’s output with 55% homology. This similarity between models generated and high homology rate gave support to being able to expect similar results from the mutations.
 
-
Further validation was found using a multiple sequence alignment between: LC-Cutinase, 3visB, Tfu_0883 and 1jfr (show pic). The alignment showed strong homology in all active site residues as well as between two of the three residues targeted by Sulaiman et. al. for mutation. The third targeted residue, though significantly different in Tfu_0883 compared to in LC-Cutinase, was expected to result in similar protein activity gains upon mutation to Alanine. This was found by loading both models for 3visB and 1jfr in Pymol and assessing that the residues held similar placement above the active site. Their placement, as well as the placement of the other two targeted residues, showed that mutation to a smaller residue, Alanine, would result in better binding ability of the active site.
 
-
</article></div>
+
<table background="transparent">
 +
<tr>
 +
<td>
 +
<a href="https://static.igem.org/mediawiki/2012/b/b5/UCD_Data_large_20.jpg" class="lightbox"><img src="https://static.igem.org/mediawiki/2012/7/73/UCD_Data_20.jpg" width="350" height="320" align="left"></a><a target="new" href="http://tcoffee.crg.cat/apps/tcoffee/do:regular"></a><br><captiontext>Multiple sequence alignment results. Positions 177 (Serine) 225 (Asparagine) 255 (Histidine) on the bottom consensus sequence mark active domains which can be seen to hold constant throughout the multiple sequences. </captiontext></td>
 +
<td>Further validation was found using a multiple sequence alignment (shown to the right) using <a target="new" href="http://tcoffee.crg.cat/apps/tcoffee/do:regular">T-Coffee</a> between: LC-Cutinase, 3visB, Tfu_0883 and 1jfr. The alignment showed strong homology in all active site residues as well as between two of the three residues targeted by Silva et. al. for mutation. The third targeted residue, though significantly different in Tfu_0883 compared to in LC-Cutinase, was expected to result in similar protein activity gains upon mutation to alanine. This reasoning was supported by loading both models for 3visB and 1jfr in Pymol and assessing that the residues held a similar placement above the active site. Their placement, as well as the placement of the other two targeted residues, also showed that mutation to a smaller residue, alanine, would result in better binding ability into the active site by the ligand.</td></div></tr></table>
 +
<br><br>
 +
 +
<p>Generating our own theoretical mutations</p>
 +
 +
<center><a href="https://static.igem.org/mediawiki/2012/1/18/UCD_Figure_large-8.jpg" class="lightbox"><img src="https://static.igem.org/mediawiki/2012/8/81/UCD_Figure-8.png "> </a> </center><br>
 +
<table background="transparent">
 +
<tr>
 +
<td>
 +
<a href="https://static.igem.org/mediawiki/2012/0/02/PET_Docking_.png" class="lightbox"><img src="https://static.igem.org/mediawiki/2012/5/58/UCD_docking_small.jpg"></a><captiontext>SwissDock results of best fit sites for PET in LC-Cutinase.</captiontext>
 +
<br><br><br>
 +
<a href="https://static.igem.org/mediawiki/2012/4/46/UCD_Data_large_19.jpg" class="lightbox"><img align="right" src="https://static.igem.org/mediawiki/2012/4/4a/UCD_Data_19.jpg"></a><captiontext>SwissDock results for ultimate best fit position of PET in LC-Cutinase</captiontext>
 +
</td>
 +
<td>
 +
After validating the previous mutations we wished to make our own. The first step was to generate a theoretical 3D model for LC-Cutinase. This was done by using Swiss-Model again to fit LC-Cutinase’s amino acid sequence into the structure of 3visB. The results, including the stability of each part of the sequence, are shown below. The results show generally high stability along the sequence. Also, upon overlapping this newly generated model of LC-Cutinase with 3visB’s model in Pymol  we received a low RMS value of .78A. This resulting data supported the idea that the generated 3D model for LC-Cutinase was stable and fit 3visB’s structure closely. The next stage was to get our theoretical model for LC-Cutinase and the two ligands it would interact with, PET and pNPB, into the <a target="new" href="http://fold.it/portal/">Foldit program</a>, a protein folding simulator. In Foldit we could perform and assay theoretical point mutations for increased stability and function of the protein. However, because Foldit does not directly measure function but measures stability instead, it was necessary to load LC-Cutinase with each ligand docked correctly into the active site. This would allow changes made at the active site that increased stability due to better ligand/protein interaction to also equate to better ligand/protein binding and thus better function by the protein. We created the ligand files through <a target="new" href="http://cactus.nci.nih.gov/translate/">Cactus</a> and the docking of the protein with each of the two ligands was done through <a target="new" href="http://swissdock.vital-it.ch/">Swissdock</a>. The images to the left show the overall docking results as well as the best fit ligand binding position. From here we loaded each specific ligand/protein combo into foldit and generated the following mutations with each rational.</td></tr></table>
 +
 +
<br><br>
 +
<p>Mutant List and Rationale</p>
 +
<ul><li><b>Silva et al. Mutant Replicants</b>
 +
<ul><li>T96A: Increase active site and change a hydrophilic residue for a hydrophobic alanine</li></ul>
 +
<ul><li>Y127A:Increase active site and change a hydrophilic residue for a hydrophobic alanine</li></ul><ul><li>V212A:Enlarge active site</li></ul></li></ul>
 +
 +
<ul><li><b>Team chosen mutants</b>
 +
<ul><li>S101A:Increase active site and change a hydrophilic residue for a hydrophobic alanine</li></ul>
 +
<ul><li>Y95A:Greatly open active site by removal of bulky side group and increase active site's hydrophobicity</li></ul>
 +
<ul><li>D98T:Enlarge active site and generated hydrogen bond</li></ul><ul><li>F125R:Enlarge active site</li></ul>
 +
<ul><li>F125Y:Enlarge active site</li></ul>
 +
<ul><li>F125A:Increase active site and change a hydrophilic residue for a hydrophobic alanine</li></ul>
 +
<ul><li>T96G:Similar as T96A however amino acid change takes place right before a alpha helix and a replacement with a G instead of A will avoid helix breaking</li></ul></li></ul>
 +
 +
 +
 +
<br><p> Additional Photos</p>
 +
<center>
 +
 +
<a href="https://static.igem.org/mediawiki/2012/5/56/UCD_Data_large_16.jpg" class="lightbox"><img align="right" src="https://static.igem.org/mediawiki/2012/4/4f/UCD_Data_16.jpg"></a>
 +
 +
<a href="https://static.igem.org/mediawiki/2012/3/3f/Homology_Model_Data_1.png" class="lightbox"><img align="left" src="https://static.igem.org/mediawiki/2012/c/cf/UCD_Data_29.jpg" ></a>
 +
</center>
 +
 +
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>1) Homology model results. Right shows overall stability across amino acid sequence. 
 +
<br><br>
 +
 +
<center>
 +
<a href="https://static.igem.org/mediawiki/2012/5/56/UCD_Data_large_18.jpg" class="lightbox"><img align="left" src="https://static.igem.org/mediawiki/2012/8/86/UCD_Data_18.jpg"></a></center>
 +
 +
<a href="https://static.igem.org/mediawiki/2012/8/80/UCD_Data_large_17.jpg" class="lightbox"><img align="right" src="https://static.igem.org/mediawiki/2012/f/ff/UCD_Data_17.jpg" width="300" height="300"></a></center>
 +
 +
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>2) PET (Yellow and red) and LC Cutinase model loaded in Foldit
 +
<br> 3) Overlapping of 3VisB and generated LC Cutinase homology models.
<br>
<br>
-
<div class="floatbox3">
+
 
-
<hp>References</hp>
+
</article>
 +
</div>
 +
 
 +
<br>
 +
<div class="smallbox" id="myleftbox"><h1> References </h1>
<article>
<article>
-
Text goes here
+
1. Silva C, et al. 2011. Engineered Thermobifida fusca cutinase with increased activity on polyester substrates. Biotechnol. J. 6:1230–1239.
-
</article></div>
+
<br>
 +
2. S. Sulaiman, S. Yamato, E. Kanaya, J. Kim, Y. Koga, K. Takano, S. Kanaya. "Isolation of a Novel Cutinase Homolog with Polyethylene Terephthalate-Degrading Activity from Leaf-Branch Compost by Using a Metagenomic Approach." Applied and Environment Microbiology, vol. 78 no. 5, pp. 1556-1562, March 2012.
 +
<br>
 +
3. Ö. Faiz et al.
 +
Determination and characterization of thermostable esterolytic activity from a novel thermophilic bacterium Anoxybacillus gonensis
 +
J. Biochem. Mol. Biol., 40 (2007), pp. 588–594<br>
 +
</article>
 +
</div>
 +
 +
 +
 +
<!-- site map starts here -->
 +
<div id="myleftbox"  class="smallboxsite">
 +
<ul style="font-size:10px;list-style-image:none;list-style-type:none;float:left;display:inline;color:#000000;"
 +
>
 +
 +
<li style="float:left;margin:0 10px;"><a
 +
href="https://2012.igem.org/Team:UC_Davis"><p>Home</p><ul
 +
style="text-indent:-15px;
 +
list-style-image:none;list-style-type:none;color:#000000;"><li><a
 +
style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis">Welcome</a> </li><li><a
 +
style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis">Tweets</a></li><li><a
 +
style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis">Sponsors</a> </li><li><a
 +
style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Criteria">Criteria</a> </li>
 +
</ul> </a> </li>
 +
 +
<li style="float:left;margin:0 10px;"><a
 +
href="https://2012.igem.org/Team:UC_Davis/Team"><p>Team</p><ul
 +
style="text-indent:-15px;
 +
list-style-image:none;list-style-type:none;color:#000000;"><li><a
 +
style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Team">Who we are</a>
 +
</li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Team">Students</a></li><li><a
 +
style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Team">Advisors</a> </li>
 +
</ul> </a> </li>
 +
 +
<li style="float:left ;margin:0 10px;"><a
 +
href="https://2012.igem.org/Team:UC_Davis/Project "><p>Project</p></a>
 +
<ul style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000"><li><a
 +
style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Project">Project Overview</a>
 +
</li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Project/Catalyst ">Module
 +
Engineering</a></li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Project/Protein_Engineering
 +
">Protein Engineering</a></li>
 +
<li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Project/Strain ">Chassis
 +
Engineering</a></li>
 +
  <li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Project/Directed_Evolution ">
 +
- Directed Evolution</a></li>
 +
  <li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Project/Our_Strain "> -
 +
Rational Engineering</a></li>
 +
<li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Criteria">Critera</a>
 +
</li></ul> </li>
 +
 +
<li style="float:left ;margin:0 10px"><a
 +
href="https://2012.igem.org/Team:UC_Davis/Safety "> <p>Safety</p></a>
 +
<a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Safety "> Safety</a> </li>
 +
 +
<li style="float:left ;margin:0 10px;"><a
 +
href="https://2012.igem.org/Team:UC_Davis/Notebook ">
 +
<p>Notebook</p></a> <ul
 +
style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000
 +
"><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Notebook">Notebook</a>
 +
</li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Notebook/Protocols
 +
">Protocols</a> </li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Notebook/Gallery">Gallery</a>
 +
</li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Criteria">Critera</a> </li>
 +
</ul> </li>
 +
 +
<li style="float:left ;margin:0 10px;"><a
 +
title="https://2012.igem.org/Team:UC_Davis/Data "> <p>Data </p></a> <ul
 +
style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000
 +
"><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Data/Cutinase_Activity ">
 +
Cutinase Activity</a> </li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol ">
 +
Ethylene Glycol</a> </li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Data/Modeling ">
 +
Modeling</a> </li><li><a style="color:#000000 "
 +
 +
href="https://2012.igem.org/Team:UC_Davis/Parts ">Parts</a></li> </ul>
 +
 +
<li style="float:left ;margin:0 10px"><a
 +
href="https://2012.igem.org/Team:UC_Davis/Attributions ">
 +
<p>Attribution </p></a><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Attributions ">
 +
Attribution</a></li>
 +
 +
<li style="float:left ;margin:0 10px"><a
 +
href="https://2012.igem.org/Main_Page "> <p>iGEM </p></a><ul
 +
style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000
 +
"><li><a style="color:#000000 " href="https://2012.igem.org/Main_Page
 +
">Main iGEM</a> </li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Criteria "> Criteria</a>
 +
</li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Human_Practices ">Human
 +
Practices</a></li> </ul>
 +
</div>
 +
<!-- site map ends here -->
 +
 +
</div>
</div>
</div>
 +
<!--  </div> -->
<script>if (window.runOnloadHook) runOnloadHook();</script>
<script>if (window.runOnloadHook) runOnloadHook();</script>
</div>
</div>
</div>
</div>
-
 
-
 
</body>
</body>
 +
<!-- footer starts here -->
 +
<br>
 +
<div id="footpage">
 +
<div class="printfooter">
 +
Retrieved from "<a href="https://2012.igem.org/Team:UC_Davis">https://2012.igem.org/Team:UC_Davis</a>"</div>
 +
<div id="catlinks"><div id='catlinks' class='catlinks catlinks-allhidden'></div></div> <!-- end content -->
 +
<div class="visualClear"></div>
 +
</div>
 +
    </div>
 +
 +
 +
<script>if (window.runOnloadHook) runOnloadHook();</script>
 +
</div>
 +
</div>
 +
<!-- footer ends here -->
</html>
</html>

Latest revision as of 02:46, 27 October 2012

Team:UC Davis - 2012.igem.org

UCDavis iGEM Tweets

Our Sponsors

Protein Engineering

Beyond the general circuit outline for our degradation of PET, there are also a protein engineering project we performed. In the project we constructed a more effective and higher yield form of the LC-Cutinase protein. We hoped to move toward a more realistic and economically feasible rate of degradation.

LC-Cutinase Engineering

Confirming expectation of common results from previous mutations

We had two goals with the engineering of cutinase: replicate mutations made in the paper by Silva et. al., which consisted of three residues mutated to alanines (1), as well as to generate our own chosen mutations. Before we decided to replicate Silva’s mutations we initially wanted to validate if we could expect the same results in our protein with the same mutations. In order to do this we wanted to compare best fit models (proteins with known structure who's sequence best matches an input sequence) generated for LC-Cutinase’s sequence and Silva’s Tfu_0883’s sequence. Replicating Silva et al. (1) we used Swiss-Model to generate a best fit model for LC-Cutinase, resulting in the protein 3visB with 61% homology. In Silva’s paper they received 1jfr, a model that was second best in LC-Cutinase’s output, with a 55% homology (1). This similarity between generated models and high homology rate gave support to the expectation of similar results from the same mutations.





Multiple sequence alignment results. Positions 177 (Serine) 225 (Asparagine) 255 (Histidine) on the bottom consensus sequence mark active domains which can be seen to hold constant throughout the multiple sequences. 		Further validation was found using a multiple sequence alignment (shown to the right) using T-Coffee between: LC-Cutinase, 3visB, Tfu_0883 and 1jfr. The alignment showed strong homology in all active site residues as well as between two of the three residues targeted by Silva et. al. for mutation. The third targeted residue, though significantly different in Tfu_0883 compared to in LC-Cutinase, was expected to result in similar protein activity gains upon mutation to alanine. This reasoning was supported by loading both models for 3visB and 1jfr in Pymol and assessing that the residues held a similar placement above the active site. Their placement, as well as the placement of the other two targeted residues, also showed that mutation to a smaller residue, alanine, would result in better binding ability into the active site by the ligand.


Generating our own theoretical mutations


SwissDock results of best fit sites for PET in LC-Cutinase.


SwissDock results for ultimate best fit position of PET in LC-Cutinase 		After validating the previous mutations we wished to make our own. The first step was to generate a theoretical 3D model for LC-Cutinase. This was done by using Swiss-Model again to fit LC-Cutinase’s amino acid sequence into the structure of 3visB. The results, including the stability of each part of the sequence, are shown below. The results show generally high stability along the sequence. Also, upon overlapping this newly generated model of LC-Cutinase with 3visB’s model in Pymol we received a low RMS value of .78A. This resulting data supported the idea that the generated 3D model for LC-Cutinase was stable and fit 3visB’s structure closely. The next stage was to get our theoretical model for LC-Cutinase and the two ligands it would interact with, PET and pNPB, into the Foldit program, a protein folding simulator. In Foldit we could perform and assay theoretical point mutations for increased stability and function of the protein. However, because Foldit does not directly measure function but measures stability instead, it was necessary to load LC-Cutinase with each ligand docked correctly into the active site. This would allow changes made at the active site that increased stability due to better ligand/protein interaction to also equate to better ligand/protein binding and thus better function by the protein. We created the ligand files through Cactus and the docking of the protein with each of the two ligands was done through Swissdock. The images to the left show the overall docking results as well as the best fit ligand binding position. From here we loaded each specific ligand/protein combo into foldit and generated the following mutations with each rational.


Mutant List and Rationale

  • Silva et al. Mutant Replicants
    • T96A: Increase active site and change a hydrophilic residue for a hydrophobic alanine
    • Y127A:Increase active site and change a hydrophilic residue for a hydrophobic alanine
    • V212A:Enlarge active site
  • Team chosen mutants
    • S101A:Increase active site and change a hydrophilic residue for a hydrophobic alanine
    • Y95A:Greatly open active site by removal of bulky side group and increase active site's hydrophobicity
    • D98T:Enlarge active site and generated hydrogen bond
    • F125R:Enlarge active site
    • F125Y:Enlarge active site
    • F125A:Increase active site and change a hydrophilic residue for a hydrophobic alanine
    • T96G:Similar as T96A however amino acid change takes place right before a alpha helix and a replacement with a G instead of A will avoid helix breaking

Additional Photos

















1) Homology model results. Right shows overall stability across amino acid sequence.

















2) PET (Yellow and red) and LC Cutinase model loaded in Foldit
3) Overlapping of 3VisB and generated LC Cutinase homology models.

References

1. Silva C, et al. 2011. Engineered Thermobifida fusca cutinase with increased activity on polyester substrates. Biotechnol. J. 6:1230–1239.
2. S. Sulaiman, S. Yamato, E. Kanaya, J. Kim, Y. Koga, K. Takano, S. Kanaya. "Isolation of a Novel Cutinase Homolog with Polyethylene Terephthalate-Degrading Activity from Leaf-Branch Compost by Using a Metagenomic Approach." Applied and Environment Microbiology, vol. 78 no. 5, pp. 1556-1562, March 2012.
3. Ö. Faiz et al. Determination and characterization of thermostable esterolytic activity from a novel thermophilic bacterium Anoxybacillus gonensis J. Biochem. Mol. Biol., 40 (2007), pp. 588–594

Retrieved from "http://2012.igem.org/Team:UC_Davis/Project/Protein_Engineering"