Team:Manchester/Enzymetest

From 2013.igem.org

(Difference between revisions)
 
(79 intermediate revisions not shown)
Line 1: Line 1:
{{:Team:Manchester/layout/noheader}}
{{:Team:Manchester/layout/noheader}}
 +
{{:Team:Manchester/Navbartest2}}
{{:Team:Manchester/Footer}}
{{:Team:Manchester/Footer}}
Line 6: Line 7:
<title> Safety </title>
<title> Safety </title>
-
<script type='text/javascript'>  
+
<script type='text/javascript'>
-
      function blocking(nr)
+
 
 +
function blocking(nr)
       {
       {
         displayNew = (document.getElementById(nr).style.display == 'none') ? 'block' : 'none';
         displayNew = (document.getElementById(nr).style.display == 'none') ? 'block' : 'none';
         document.getElementById(nr).style.display = displayNew;
         document.getElementById(nr).style.display = displayNew;
       }   
       }   
-
function hover1()
 
-
{
 
-
  $("#block").hover(function() {
 
-
      $("#block #image1").toggleClass("expand");
 
-
  });
 
-
  $("#block").mouseenter(function() {
 
-
    $("#block #image1").css({marginLeft: "40px"});
 
-
  });
 
-
  $("#block").mouseleave(function() {
 
-
    $("#block #image1").css({marginLeft: "50px"});
 
-
  });
 
-
}
 
-
function hover2()
 
-
{
 
-
  $("#block").mouseenter(function() {
 
-
    $("#block #image1").css({marginLeft: "40px"});
 
-
  });
 
-
    $("#block").mouseleave(function() {
 
-
    $("#block #image1").css({marginLeft: "50px"});
 
-
  });
 
-
}
 
-
function hover3()
 
-
{
 
-
  $("#block1").hover(function() {
 
-
      $("#block1 #image2").toggleClass("expand");
 
-
  });
 
-
  $("#block1").mouseenter(function() {
 
-
    $("#block1 #image2").css({marginLeft: "40px"});
 
-
  });
 
-
  $("#block1").mouseleave(function() {
 
-
    $("#block1 #image2").css({marginLeft: "50px"});
 
-
  });
 
-
  $("#block1").mouseenter(function() {
 
-
      $("#block1 .submenu").show();
 
-
  });
 
-
  $("#block1").mouseleave(function() {
 
-
      $("#block1 .submenu").hide();
 
-
  });
 
-
}
 
-
 
-
function hover4()
 
-
{
 
-
  $("#block2").hover(function() {
 
-
      $("#block2 #image3").toggleClass("expand");
 
-
  });
 
-
  $("#block2").mouseenter(function() {
 
-
    $("#block2 #image3").css({marginLeft: "40px"});
 
-
  });
 
-
  $("#block2").mouseleave(function() {
 
-
    $("#block2 #image3").css({marginLeft: "50px"});
 
-
  });
 
-
  $("#block2").mouseenter(function() {
 
-
      $("#block2 .submenu").show();
 
-
  });
 
-
  $("#block2").mouseleave(function() {
 
-
      $("#block2 .submenu").hide();
 
-
  });
 
-
}
 
-
 
-
 
-
function hover5()
 
-
{
 
-
  $("#block3").hover(function() {
 
-
      $("#block3 #image6").toggleClass("expand");
 
-
  });
 
-
  $("#block3").mouseenter(function() {
 
-
    $("#block3 #image6").css({marginLeft: "40px"});
 
-
  });
 
-
  $("#block3").mouseleave(function() {
 
-
    $("#block3 #image6").css({marginLeft: "50px"});
 
-
  });
 
-
  $("#block3").mouseenter(function() {
 
-
      $("#block3 .submenu").show();
 
-
  });
 
-
  $("#block3").mouseleave(function() {
 
-
      $("#block3 .submenu").hide();
 
-
  });
 
-
}
 
-
 
-
function hover6()
 
-
{
 
-
  $("#block4").hover(function() {
 
-
      $("#block4 #image4").toggleClass("expand");
 
-
  });
 
-
  $("#block4").mouseenter(function() {
 
-
    $("#block4 #image4").css({marginLeft: "40px"});
 
-
  });
 
-
  $("#block4").mouseleave(function() {
 
-
    $("#block4 #image4").css({marginLeft: "50px"});
 
-
  });
 
-
  $("#block4").mouseenter(function() {
 
-
      $("#block4 .submenu").show();
 
-
  });
 
-
  $("#block4").mouseleave(function() {
 
-
      $("#block4 .submenu").hide();
 
-
  });
 
-
}
 
-
 
-
 
-
function hover7()
 
-
{
 
-
  $("#block5").hover(function() {
 
-
      $("#block5 #image5").toggleClass("expand");
 
-
  });
 
-
  $("#block5").mouseenter(function() {
 
-
    $("#block5 #image5").css({marginLeft: "40px"});
 
-
  });
 
-
  $("#block5").mouseleave(function() {
 
-
    $("#block5 #image5").css({marginLeft: "50px"});
 
-
  });
 
-
  $("#block5").mouseenter(function() {
 
-
      $("#block5 .submenu").show();
 
-
  });
 
-
  $("#block5").mouseleave(function() {
 
-
      $("#block5 .submenu").hide();
 
-
  });
 
-
}
 
</script>
</script>
Line 153: Line 38:
-webkit-border-radius: 10px;
-webkit-border-radius: 10px;
border-radius: 10px;  
border-radius: 10px;  
-
-webkit-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
+
 
-
-moz-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
+
-webkit-box-shadow: 0px 0px 5px 0px rgba(76,0,130,1);
-
box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
+
-moz-box-shadow: 0px 0px 5px 0px rgba(76,0,130,1));
 +
box-shadow: 0px 0px 5px 0px rgba(76,0,130,1);
}
}
Line 184: Line 70:
{
{
float:left;
float:left;
-
margin-left:110px;
+
margin-left:90px;
-
margin-right:20px;
+
margin-right:10px;
}
}
Line 192: Line 78:
float:left;
float:left;
margin-left:50px;
margin-left:50px;
 +
}
 +
 +
.global
 +
{
 +
position:relative;
 +
width:950px;
 +
height:1600px;
}
}
Line 202: Line 95:
left:0;
left:0;
padding:10px;
padding:10px;
-
background-color:#f2f2f2;
+
background-color:white;
-webkit-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
-webkit-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
Line 209: Line 102:
}
}
-
.container
+
.wrapper2
{
{
-
height:90px;
 
-
width:980px;
 
-
margin:0 auto;
 
-
background-color:#F2F2F2;
 
position:absolute;
position:absolute;
-
top:20px;
+
clear:both;
 +
width:940px;
 +
top:120px;
left:0;
left:0;
-
z-index:100;
+
padding:10px;
-
}
+
background-color:#f2f2f2;
-
#block, #block1, #block2, #block3, #block4, #block5
+
-webkit-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
 +
-moz-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
 +
box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
 +
}
 +
.menu
{
{
-
float:left;
+
margin:5px auto;
-
width:150px;
+
width:900px;
-
height:80px;
+
-
margin:0 5px;
+
}
}
-
#block a, #block1 a, #block2 a, #block3 a, #block4 a, #block5 a
+
.menu li
{
{
-
display:block;
+
list-style:none;
-
text-decoration: none;
+
-
background-color: #660099;
+
-
color:white;
+
-
font-size: 17px;
+
-
font-family: Trebuchet MS;
+
-
font-weight: bold;
+
-
border-radius: 5px;
+
-
width: 140px;
+
-
margin:0 auto;
+
-
text-align: center;
+
-
padding: 3px;
+
-
color:#F2F2F2;
+
-
border:1px solid #4C0082;
+
-
 
-
-webkit-transition-duration:0.1s;
 
-
-webkit-timing-function:linear;
 
-
transition-duration:0.1s;
 
-
transition-timing-function:linear;
 
}
}
-
#block img,#block1 img,#block2 img,#block3 img,#block4 img, #block5 img
+
.menu li #mlink
{
{
-
-webkit-transition-duration:0.1s;
 
-
-webkit-timing-function:linear;
 
-
transition-duration:0.1s;
 
-
transition-timing-function:linear;
 
-
margin:0 50px;
 
display:block;
display:block;
-
}
+
width:900px;
 +
text-decoration:none;
 +
margin-bottom:5px;
 +
font-family:Trebuchet MS;
 +
font-weight:bold;
 +
font-size:20px;
 +
color:white;
 +
background-color:#660099;
 +
padding:7px 5px 5px 5px;
-
#block img:hover,#block1 img:hover,#block2 img:hover,#block3 img:hover,#block4 img:hover, #block5 img:hover
+
-webkit-border-radius: 10px;
-
{
+
border-radius: 10px;  
-
width:65px;
+
-
height:65px;
+
-
}
+
-
#block .list,#block1 .list,#block2 .list,#block3 .list,#block4 .list
+
-webkit-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
-
{
+
-moz-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
-
margin:0;
+
box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
}
}
-
#block .list li, #block1 .list li, #block2 .list li, #block3 .list li, #block4 .list li
+
.menu li a #date
{
{
-
display:block;
+
margin-left:10px;
-
list-style:none;
+
margin-right:10px;
-
margin:0;
+
}
}
-
.submenu
+
.menu li a #arrow
{
{
-
position:relative;
+
margin-left:10px;
-
display:none;
+
-
clear: both;
+
-
margin:0;
+
-
display: none;
+
}
}
-
#block .list .submenu li
+
#moretext
{
{
-
list-style:none;
 
-
display: block;
 
-
margin:0;
 
-
float: none;
 
-
}
 
-
 
-
#block1 .list .submenu li a, #block2 .list .submenu li a, #block3 .list .submenu li a, #block4 .list .submenu li a
 
-
{
 
-
clear:both;
 
-
display:block;
 
-
border:1px solid #4C0082;
 
margin:0 auto;
margin:0 auto;
-
width:130px;
+
margin-bottom:5px;
-
font-size: 13px;
+
width:700px;
-
font-family: Trebuchet MS;
+
font-family:Trebuchet MS;
-
font-weight: bold;
+
font-weight:bold;
-
color:#f2f2f2;
+
font-size:14px;
-
}
+
color:white;
 +
background-color:#BDBDBD;
 +
padding:5px;
-
#block4 .list .submenu li .submenu1, #block4 .list .submenu li .submenu2
+
-webkit-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
-
{
+
-moz-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
-
margin:0;
+
box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
-
}
+
-
+
-
#block4 .list .submenu li .submenu1 a, #block4 .list .submenu li .submenu2 a
+
-
{
+
-
margin:0 auto;
+
-
width:110px;
+
-
font-size: 13px;
+
-
font-family: Trebuchet MS;
+
-
font-weight: bold;
+
-
color:black;
+
-
background-color:#FFCC33;
+
}
}
-
#block4 .list .submenu li .submenu1 a:hover, #block4 .list .submenu li .submenu2 a:hover
+
.container1
{
{
-
color:white;
+
margin:10px;
-
display:block;
+
width:900px;
-
background:#A901DB;
+
height:320px;
 +
padding:10px;
 +
background-color:#F2F2F2;
 +
-webkit-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
 +
-moz-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
 +
box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
}
}
-
#block1 .list .submenu li a:hover, #block2 .list .submenu li a:hover, #block2 .list .submenu li a:hover,
+
.container1 img
-
#block3 .list .submenu li a:hover, #block4 .list .submenu li a:hover
+
{
{
-
display:block;
+
-webkit-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
-
background:#A901DB;
+
-moz-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
 +
box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
}
}
-
#block .list li:hover .submenu
+
.container1 #title
{
{
-
display:block;
+
float:left;
 +
margin:10px;
}
}
-
.expand
+
.container1 #pic
{
{
-
-webkit-transition-duration:0.1s;
+
float:right;
-
-webkit-timing-function:linear;
+
margin:10px;
-
transition-duration:0.1s;
+
-
transition-timing-function:linear;
+
-
 
+
-
width:65px;
+
-
height:65px;
+
}
}
-
.menu
+
.text1
{
{
-
margin:5px auto;
+
margin:10px;
width:900px;
width:900px;
 +
font-style:Trebuchet MS;
 +
font-size:14px;
 +
color:#4c0082;
 +
background-color:#F2F2F2;
 +
padding:10px;
 +
 +
-webkit-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
 +
-moz-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
 +
box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
}
}
-
.menu li
+
.text1 p
{
{
-
list-style:none;
+
font-style:Trebuchet MS;
 +
font-size:14px;
 +
color:#4c0082;
 +
background-color:#F2F2F2;
}
}
-
.menu li a
+
.text1 #footer
{
{
-
display:block;
+
float:right;
-
width:900px;
+
width:500px;
-
text-decoration:none;
+
margin:0 10px 5px 10px;  
-
margin-bottom:5px;
+
-
font-family:Trebuchet MS;
+
-
font-weight:bold;
+
-
font-size:24px;
+
-
color:white;
+
-
background-color:#660099;
+
-
padding:5px;
+
-
 
+
-
-webkit-border-radius: 10px;
+
-
border-radius: 10px;  
+
-webkit-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
-webkit-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
Line 389: Line 242:
}
}
-
center img
+
.text1 img
{
{
-
margin:10px auto;
+
float:right;
 +
margin:10px;
-webkit-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
-webkit-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
-moz-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
-moz-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
Line 397: Line 251:
}
}
-
.menu #month0 a span
+
.text2
{
{
-
margin-left:20px;
+
margin:10px;
-
margin-right:330px;
+
width:900px;
 +
font-style:Trebuchet MS;
 +
font-size:14px;
 +
color:#4c0082;
 +
background-color:#F2F2F2;
 +
padding:10px;
 +
 
 +
-webkit-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
 +
-moz-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
 +
box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
}
}
-
.menu #month1 a span, .menu #month2 a span
+
.text2 #footer
{
{
-
margin-left:20px;
+
float:left;
-
margin-right:370px;
+
width:450px;
 +
margin:0 10px 5px 10px;
 +
 
 +
-webkit-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
 +
-moz-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
 +
box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
}
}
-
.menu #month3 a span
+
.text2 p
{
{
-
margin-left:20px;
+
font-style:Trebuchet MS;
-
margin-right:360px;
+
font-size:14px;
 +
color:#4c0082;
 +
background-color:#F2F2F2;
}
}
-
.menu #month4 a span
+
.text2 img
{
{
-
margin-left:20px;
+
float:left;
-
margin-right:340px;
+
margin:10px;
 +
-webkit-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
 +
-moz-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
 +
box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
}
}
-
#submenu101 li a, #submenu li a, #submenu2 li a, #submenu3 li a, #submenu4 li a
+
.text3
{
{
-
display:block;
+
margin:10px;
-
width:800px;
+
width:900px;
-
margin-left:30px;
+
font-style:Trebuchet MS;
-
padding:5px;
+
font-size:14px;
-
text-align:center;
+
color:#4c0082;
-
font-family:Trebuchet MS;
+
background-color:#F2F2F2;
-
font-weight:bold;
+
padding:10px;
-
font-size:18px;
+
-
color:white;
+
-
background-color:#FFCC33;
+
-webkit-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
-webkit-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
Line 439: Line 309:
}
}
-
#text101, #text102, #text103,  #text, #text1, #text2, #text3,#text4,#text5,#text6,#text7,#text8,#text9,#text10,#text11,#text12,#text13,#text14,#text15
+
.text3 #footer
{
{
-
margin:0 auto;
+
width:890px;
-
margin-bottom:5px;
+
margin-top:5px;  
-
width:700px;
+
-
font-family:Trebuchet MS;
+
-
font-weight:bold;
+
-
font-size:14px;
+
-
color:white;
+
-
background-color:#BDBDBD;
+
padding:5px;
padding:5px;
Line 456: Line 320:
}
}
-
.global
+
.text3 img
{
{
-
position:relative;
+
-webkit-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
-
width:950px;
+
-moz-box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
-
height:1600px;
+
box-shadow: 0px 0px 5px 0px rgba(0,0,0,0.75);
}
}
 +
 +
.block1 a, .block2 a, .block3 a, .block4 a, .block5 a, .block6 a, .block7 a, .block8 a, .block9 a, .block10 a, .block11 a, .block12 a, .block13 a
.block1 a, .block2 a, .block3 a, .block4 a, .block5 a, .block6 a, .block7 a, .block8 a, .block9 a, .block10 a, .block11 a, .block12 a, .block13 a
Line 502: Line 368:
.block2 a
.block2 a
{
{
 +
display:block;
display:block;
float:left;
float:left;
Line 519: Line 386:
.block4 a
.block4 a
{
{
 +
display:block;
display:block;
float:left;
float:left;
Line 527: Line 395:
.block5 a
.block5 a
{
{
 +
display:block;
display:block;
float:left;
float:left;
Line 541: Line 410:
}
}
-
.block7 a
+
.rightbar
{
{
-
display:block;
+
width:120px;
 +
position:fixed;
 +
top:120px;
 +
right:15px;
 +
}
 +
 
 +
.question1 a, .question2 a, .question3 a, .question4 a, .question5 a
 +
{
 +
width:120px;
 +
text-decoration:none;
 +
color:white;
 +
text-align:center;
 +
background:#606060;
 +
padding:5px;
 +
font-style:Trebuchet MS;
 +
font-weight:bold;
 +
font-size:12px;
 +
color:white;
 +
}
 +
 
 +
.question1 a:hover, .question2 a:hover, .question3 a:hover, .question4 a:hover, .question5 a:hover
 +
{
 +
background:#C0C0C0;
 +
color:#606060;
 +
}
 +
 
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<li id="one"><a id="mlink" href="" onclick="blocking('text1'); return false;"><span id="arrow"><img src="https://static.igem.org/mediawiki/2013/e/ee/DownArrow2.png" width="20" height="20"/></span> Table of all parameters</a>
 
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<iframe width='700' height='1200' frameborder='0' src='https://docs.google.com/spreadsheet/pub?key=0Ajbiu1uO_n5xdFFwdVptNFN0NXdHcDlIeFFrUjVRRmc&single=true&gid=0&output=html&widget=true'></iframe>
 
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                <li id="one"><a href="" onclick="blocking('text102'); return false;">Public Outreach Planning</a>
 
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-
<p>From the start of the project we had decided that we would include outreach activities aimed at young people in order to interest and educate them, but also to promote the field of synthetic biology to the next generation. Luckily, Elsa had some contacts within the Faculty of Life Sciences (FLS) at the university, and we quickly managed to sign up for both a two-day workshop event and the annual Community Open Day! (More info can be found on our Public Outreach pages)</p>
 
-
<br>
 
-
<p>Having received word of a university-wide competition to encourage public engagement, Jess gave a presentation to the Head of Public Engagement within FLS and to the other competitors, ultimately winning us £150 to go towards our outreach events and an Outreach Mentor (Matthew Hickman)! We then met with Matthew and discussed our activity ideas, and he gave us lots of useful hints and tips of the dos and don’ts associated with hosting outreach events.</p>
 
-
<br>
 
-
<p>After much deliberation we finally settled on what our main activity would be - a hands-on workshop where the children would build a DNA double helix out of sweets (representing the base pairs), strawberry pencils (representing the sugar-phosphate backbone) and cocktail sticks (representing the hydrogen bonding)! We also decided to include a mini discussion/debate amongst the children on the ethics of synthetic biology.</p>
 
-
<br>
 
-
<p>During the weeks leading up to summer (and our planned events!), the outreach team designed a poster for the Community Open Day and made a thorough plan of how our workshops would be run. The aim of the poster was to attractively present our project, the palm oil industry and the iGEM competition to a wide range of people (the Open Day was free for anyone to attend), which we certainly did!</p>
 
-
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                        </li>
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<p> <b> <u> Summary </p> </u> </b>
 +
<p>Working with a pathway as large and uncharacterised as the fatty acid biosynthesis presented many challenges, the most important of which was the lack of reliable, experimentally established kinetic values for many of the key reactions. Our solution was to create a model that explicitly acknowledges this lack of data and the resulting uncertainty, using Monte Carlo sampling from plausible parameter value distributions -- enabling us to produce <b>model predictions with confidence intervals</b>. We believe that this <b>unusual and innovative modelling strategy</b> can potentially serve as a general principled approach to handling parameter uncertainty in the future. Synthetic Biology will always operate at the cutting edge of current knowledge and thus will unavoidably face the challenge of uncertainty. Building models with incorporated acknowledgment of uncertainty will yield model predictions with specified confidence intervals, and thus will lead to more robust design strategies for a wide range of engineered cellular machines. </p>
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      <li id="month1"><a href="" onclick="blocking('submenu'); return false;"><span><img src="https://static.igem.org/mediawiki/2013/e/ee/DownArrow2.png" width="30" height="30" /></span>June</a>
+
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                  <ul id="submenu">      
+
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        <li id="one"><a href="" onclick="blocking('text'); return false;">Week 1</a>
+
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<div id="text">
+
-
                            <p>Week 1 in the Manchester iGEM house...
+
-
Exams are over and we’ve officially started full-time work on the project! At the start of the week we had a big group meeting, complete with instructors and advisors, and were presented with our very own pins and stickers set. The pins quickly vanished, but we held on to the stickers for an Outreach activity we have in the pipeline (more on that on the Outreach page!). We took advantage of the rare sunshine and had our first (almost) group photo taken, to go in an article in the uni newsletter (and to also reveal what Team Manchester looks like to the rest of the world). We were introduced to our beautiful lab space, and are all really eager to get our hands/gloves dirty!  </p>
+
-
<p>The wiki designers have begun tweaking the page to make it a little more presentable for the masses and, apart from a little accident where Ali’s face was pasted over everyone else’s on the Team page, it’s going quite swimmingly!</p>
+
          </div>
-
<p>After quickly realising that biology is SO COMPLEX, the main bulk of the team began an intensive literature search, looking for anything and everything that may be of use to us in the following weeks. We’ve located a BioBrick that we would really love to use and attempt to improve, but are having doubts about whether or not it is available for us to order. Hope so, or it’s back to square one! Fingers crossed.</p>
 
-
<p>Preliminary modelling research has also begun. None of the team has firsthand experience of modelling biology, so this should be interesting. We’re currently scouring the internet and some 1970s handbooks in search of kinetic information for the enzymes involved in prokaryotic fatty acid biosynthesis. One small step for man and all that. Wish us luck!</p>
+
<div class="text3">
 +
            <p> <u> <b> Aim </u> </b> </p>
 +
<p> <b> To use uncertainty modelling to model <i>E. coli</i> fatty acid biosynthesis.</p> </b>
-
<p> <center><img src="https://static.igem.org/mediawiki/2013/a/a6/Team1.jpg"  /></center> </p>
+
<p> Early modelling attempts using traditional methods of modelling were largely unsuccessful, due to the the nature of the fatty acid biosynthesis pathway, and the lack of experimentally defined kinetic values. Rather than use models that were arbitrary or lacked information, we decided to use a less traditional method, based on Monte Carlo sampling, that can give us a clear idea of what the uncertainty of our predictions might be. By embracing this uncertainty, we hoped to create a model with practical, representative results. </p>
-
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            <li id="two"><a href="" onclick="blocking('text1'); return false;">Week 2</a>
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-
                              <p> Week 2 in the Manchester iGEM house... (that’s old now, I won’t use it again.)
+
-
This has been a busy week for the less sciencey aspects of the project. We finalised our team logo, made a great poster for a Community open day we will be attending in July, and made the final changes to the sponsor packet we will be sending out soon. The team attempted to split into two subteams: core experimental team and core modelling team. Despite working on these two separate things, the group still managed to come together regularly to update the others on any progress made. Both experimental and modelling research started in Week 1 was built upon, and we feel that we are at least going in the right direction! </p>
+
-
<p>We put a request in for 3 BioBricks from the Repository, and are eagerly awaiting a response. Lab work will start soon. To begin with it will just be building up a stock of media and plates etc, but it will make the lab feel more like home. Which is good, because we plan on living there for the next 10 weeks.</p>
+
            <p> <u> <b> Objectives </u> </b> </p>
 +
<p>  <ul>
 +
<li> To build the first kinetic model of fatty acid biosynthesis in <i>E. coli</i> using uncertainty modeling </li>
 +
<li> To represent the fatty acid production in our system: <i>E.c(oil)i</i> </li>
 +
<li> To identify areas of the pathway requiring further study in the lab </li>
 +
<li> To develop a method of modelling that can be used in the absence of a fully characterised pathway </li>
 +
</ul>  </p>  
-
<p>We also met with Dr. Andy Balmer to have a chat about Human Practices. There was so much we hadn’t thought about! He definitely gave us a few things to consider, and we set a date for another meeting in Week 3. Following the meeting we thought it would be a good idea to appoint a Head of Human Practices and a Head of Ethics on the team, which fell to Rob and Tan respectively (congrats on your new job, here is a cake).</p>
+
          </div>
-
<p>5 of the team will be visiting London in July for the first ever Young Synthetic Biologists meet-up! Accommodation and travel has already been paid, so now it’s just a matter of waiting for the day to come. We’ll be there with bells on (and also with a soil sample, at Norwich iGEM’s request!)</p>
 
-
<p> <center><img src="https://static.igem.org/mediawiki/2013/e/e5/MancPlates.jpg" /></center> </p>
+
<div class="text3">
-
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+
            <p> <b> <u> Introduction </p> </b> </u>
-
</li>
+
<p> Fatty acid biosynthesis is a process that occurs in all living organisms. Glucose is converted into acetyl-CoA through the citric acid cycle, which is fed into the fatty acid biosynthesis pathway. Here it reacts with malonyl-CoA to form a four carbon compound. The four carbon compound is then reduced and dehydrated via four successive steps, executed, with the help of NADPH, by the enzymes as indicated in Figure 1. To this resulting C4 body, another malonyl-CoA reacts to form a C6 body - which is converted the same manner as the previous C4 body. A number of unchanging enzymes act on the intermediates of this cyclic pathway to ultimately produce fatty acids. From the initial reaction to the end products the whole pathway numbers <b> 43 reactions, about 60 metabolites and 267 parameters. </b> </p>
-
        <li id="three"><a href="" onclick="blocking('text2'); return false;">Week 3</a>
+
-
    <div id="text2">
+
-
                              <p>This week we started thinking about our FadD knockout. After a meeting about primer design with our new supervisor, Jay; we found the gene sequence, worked out our primers and put them to order! Later in the week we had another meeting with Andy Balmer, which once again gave us a lot to think about. We’ve had a few lightbulb moments and we’re pretty excited to get stuck in! Back in the lab we found out that we didn’t have any supplies, but after a quick shopping trip we ended up with plenty of supplies...ok a LOT of supplies (Not sure if we’re going to get through 3000 1.5ml Eppendorf tubes). </p>
+
-
<p>Unfortunately, our first attempt at transformation failed, but to raise our spirits we had our first iGEM social! Divita cooked the team a fantastic curry, and we had a great time. The food adventures continued the next day when Elsa brought in some Icelandic dried fish: certainly something different! </p>
+
<img src="https://static.igem.org/mediawiki/2013/2/21/Fabcyclefixed.png" width="900" height="400"/>
 +
<p id="footer"><b>Figure 1: Fatty Acid Biosynthesis Pathway, thioesterase reaction (tesA) and Δ 9, Δ12 desaturase reactions.
 +
</b></p>
-
<p>Meanwhile all through the week, the modelling team have been doing a great job making the experimental team look bad. They’ve been learning how to use Copasi (made here in Manchester!) and are cross-referencing the data ready to start simulation. </p>
+
<p> In synthetic biology two main classes of computational models are commonly used: constraint-based genome-scale models and differential-equation-based dynamic models. In our project, we were interested in the concentrations of compounds and their dynamic changes as well as the reactions with the highest control over the fatty acid synthesis pathway. As our analysis would not be possible with a purely constraint-based model, we chose to use a dynamic model. However, to use a dynamic model one needs to know the enzyme kinetic parameters, and these are often unknown or very unreliable for enzymes. Uncertainty can be due to:
 +
<li> experimental uncertainty</li>
 +
<li><i>in vitro</i> measurements of enzyme kinetics are not always representative of <i>in vivo</i> conditions</li>
 +
<li> compound concentrations often have dynamic changes </li>
 +
<b> We wanted to account for the uncertainty in the fatty acid synthesis pathway parameter data by using a new “uncertainty modelling” approach, which can potentially serve as a principled approach to handling parameter uncertainty in the future. </b> </p>
-
<p> <center><img src="https://static.igem.org/mediawiki/2013/9/95/MancShop.jpg"  /></center> </p>
+
<p> Building models with incorporated acknowledgment of uncertainty will produce specified confidence intervals for all model predictions and thus could lead to robust design of engineered cellular machines of fatty acid synthesis and beyond.
-
</div>
+
</p>
-
</li>
+
          </div>
-
        <li id="four"><a href="" onclick="blocking('text3'); return false;">Week 4</a>
+
-
    <div id="text3">
+
-
                            <p> Week 4 started off slowly, but by the end things were really starting to come together. We realised that the pkd46 plasmid we were trying to transform is heat sensitive, so heat-shocking and incubating at 37C really wasn’t the best idea! Instead we tried electroporation, which we managed to do first time, with some successful colonies! Hopefully the rest of the fadD knockout will now run smoothly. </p>
+
-
<p>After realising just how expensive biology can be, we’ve started to make a big push on the sponsorship front. So far this has led to some free kits from QIAGEN, so we're off to a good start. </p>
+
<div class="text3">
 +
<p> <b> <u> Method </p> </b> </u>
 +
<img src="https://static.igem.org/mediawiki/2013/7/7b/Workflowmod.png" width="900"/>
 +
<p id="footer"><b>Figure 2: Schematic workflow representation for building the dynamic uncertainty model split in 5 successive steps. See text for details.  
 +
</b></p>
-
<p>Unfortunately this week we also had to say goodbye to Ali and Johanna, who need to spend time on other projects. Luckily we can say hello to Marco (who will be helping in the lab) and our new advisor Denis (who’s will be helping us out with implementing our wiki ideas). </p>
+
<p> <b> <li type="1">Collected parameter values from literature</li></b>
 +
Using the online database BRENDA, we searched for published parameter values for every enzyme in the pathway. In our search we discovered that the published data on the value of parameters in the <i>E. coli</i> fatty acid biosynthesis pathway is limited. Hence, we decided we needed to take this into account in our model.<br><br>
 +
<b><li type="1">Categorized parameters into three groups </li></b>
 +
<b>Group 1:</b> ☑Mean ☑Standard Deviation<br>
 +
<b>Group 2:</b> ☑Mean ☐Standard Deviation<br>
 +
<b>Group 3:</b> ☐Mean ☐Standard Deviation<br><br>
 +
<b><li type="1"> Filling in the missing information for each parameter</li></b>
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In the case of group one, both the mean and standard deviation were collected from the literature and used to determine the probability distribution. In the case of group two, we used the mean and standard deviation of all enzymes of the same class or subclass with known kinetic parameters. In the case of group three, we used the standard deviation obtained from all enzymes of the same sub-class to create a distribution. <a href="https://static.igem.org/mediawiki/2013/d/db/Manchester_Probability_Distributions.pdf" target="_blank">Here is a file with the distributions of enzyme classes and subclasses.</a> Below is a spreadsheet of each parameter value. The source of each parameter value is hyperlinked in blue. <br><br>
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<iframe width='900' height='900' frameborder='0' src='https://docs.google.com/spreadsheet/pub?key=0Ajbiu1uO_n5xdFFwdVptNFN0NXdHcDlIeFFrUjVRRmc&output=html&widget=true'></iframe><br><br>
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<b><li type="1">Random sampling values from each parameter distribution</li></b>
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Once each parameter had a probability distribution associated with it, we randomly sampled values from each parameter distribution to run our model simulation. This was done by constructing an initial model in Copasi, using appropriate enzyme kinetic equations. The rate equations used in our model can be generalised as follows: <br><br>
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<center><img src="https://static.igem.org/mediawiki/2013/e/e0/Manre1.png" width="600" height="800"/></center>
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<center><img src="https://static.igem.org/mediawiki/2013/5/5b/Manre2.png" width="600" height="250"/></center><br><br>
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We used these rate equations to complete our model of the fatty acid synthesis pathway by adding the thioesterase reactions required for the production of the final fatty acids as well as the reactions catalyzed by FadD. Once our model was complete enough to represent our system we exported this from COPASI in SBML format and converted it to a PySCeS compatible file. PySCeS uses a set of non-linear differential equations are used to obtain both structural and kinetic information about the system from these randomly generated kinetic values. Below is pseudocode of our workflow, the <a href="https://static.igem.org/mediawiki/2013/f/ff/ManchesterModellingScript.txt" target="_blank">full script to randomly sample the parameters and generate model predictions can be downloaded here.</a><br><br>
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<center><img src="https://static.igem.org/mediawiki/2013/3/3a/Manpc.png" width="600" height="600"/></center><br><br>
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<b><li type="1">Creation of 1,000 models with distribution of predictions </li></b>
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We automated the generation of models to create a collection of 1,000 models. From there we were able to determine the uncertainty in our model predictions: instead of a single prediction, we have a distribution of predictions from a large collection of plausible models. </p>
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<p>As you can see it’s been a fairly quiet week, but with our upcoming public outreach events and YSB 1.0, we’ve got a busy fortnight ahead of us!</p>
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</div>
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 +
<div class="text3">
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<p> <b> <u> Results </p> </u> </b>
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<p> <center><img src="https://static.igem.org/mediawiki/2013/a/a0/DivRalf.jpg" /></center> </p>
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<center><img src="https://static.igem.org/mediawiki/2013/c/c1/ColourfulSpreadsheet.png" width="900" height="500"/></center>
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<p id="footer"><b> Figure 3: Short excerpt of metabolite concentration of fatty acid biosynthesis from multiple simulations within 100 seconds at ten different time points. Colours visualise concentrations according to their amount (<b>Dark Red:</b> >4; <b>Pink:</b> > 2; <b>Light red:</b>> 1; <b>White:</b> 1-0.01; <b>Light yellow:</b> <0.01; <b>Dark yellow: </b><0.0001)
 +
</b></p>
 +
<br>
 +
<p>The concentrations of the metabolites was outputted in tables, as depicted in Figure 3. Each line represents one simulation with ten different time points within 100 seconds. The whole data set of all simulations was then attributed with colours according concentration values. Another table was generated out of this chart according to the ordinal data obtained from colouring the metabolite concentrations. This was done to further improve ease of work and making the data more visual. Figure 4 shows the summary of this qualitative concentration distribution for each metabolite. Again, the brighter green a cell is in colour, the more often simulations rendered metabolite concentrations in the specified concentration interval. For example, the last metabolite in the table C18CoA is bright green, because all 41 simulations rendered between 0.01 - 1 mM. Out of this table, a clear distribution becomes obvious: Except for the first six initial replenishing reactions, all metabolite concentrations are within a small reasonable range mostly between 0.01 - 1 mM. Interestingly, in the reaction towards the end of the pathway, which are responsible for removing the metabolites from the system and therefore give rise to stearic and palmitic acid (our desired products) the range of results appears to be significantly narrower, despite the uncertainty.</p>
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</div>
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<p> The analysis of the data shows clearly, that due to a small and reasonable range of metabolite concentrations which stabilises towards the end  of the model, a high validity of our functioning model can be safely assumed and demonstrates that the uncertainty is not globally deleterious. Even though the model was working with high uncertainties in data, the output is always within a valid range.  </p>
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</li>
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<center><img src="https://static.igem.org/mediawiki/2013/d/dc/Greenspreadsheet.png" width="639" height="721"/></center>
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  </ul>
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<p id="footer"><b>Figure 4: Comprehensive summary table of all analysed models with colour coded visualisation according to qualitative concentration distribution for each metabolite. The brighter in colour a cell is, the more often the simulations resulted in the specified concentration interval. 
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  </li>  
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</b></p>
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      <li id="month2"><a href="" onclick="blocking('submenu2'); return false;"><span><img src="https://static.igem.org/mediawiki/2013/e/ee/DownArrow2.png" width="30" height="30" /></span>July</a>
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        <li id="one"><a href="" onclick="blocking('text4'); return false;">Week 5</a>
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<div id="text4">
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                              <p> This week has certainly been a mix of ups and downs! On Tuesday and Wednesday the outreach team ran 12 workshops for children aged 11-13 at the university’s Science Stars event. They taught the children about the structure of DNA by getting them to create a DNA molecule from Mario sweets (Mario pairs to Yoshi, Donkey Kong to Diddy Kong) and strawberry pencils. The activity was received amazingly well by the children and teachers alike. We also taught them about what synthetic biology is, and asked them to think what ideas they’d have as well as the ethical implications associated with them. Some of the ideas were phenomenal, and some were let’s say...a little more abstract. The team is doing the workshop again at the Community Open Day on Saturday, where they’ll be bringing SynBio to the wider public!</p>
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<p>Unfortunately, experimental progress has been plagued by admin issues, delaying us from getting the FAS module and our primers. On the plus side our electrocompetent bacterial colonies were successful, meaning our Court-Lamba recombinase knockout of the fadD gene is ready for the next step! Now we just need our primers. Modelling have had a bit more success, but both subteams have had issues with finding the source for delta-9-desaturase. Fortunately, this was more or less resolved by the end of the week!</p>
 
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<p>We also had another social! Elsa cooked the team a delicious vegetarian lasagna, we found out more about Jess’ love of rodents, and Rob tried (and failed) to do Bhangra dancing. </p>
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<p> Upon analysing the degree of certainty in our model, and finding that it was at a level that we believe is suitable for further analysis, we were able to create a series of boxplots showing the range of values found within our simulations for species accumulation after 100 seconds. We focused on the longer chain fatty acids, which are the engineering target of our pathway. The order in which the species are shown in the box plots, Figure 5, is also the order in which they are formed. This is also shown in Figure 6, where the colour corresponds to the colour of the bar on the box plot. </p>
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<center><img src="https://static.igem.org/mediawiki/2013/1/19/Boxplot.jpg" width="563" height="504"/></center>
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<p id="footer"><b>Figure 5: Summarized boxplots of the last metabolite concentrations (mM) in the modelled fatty acid synthesis pathway. Indicated colours correspond to the colours and metabolites as shown in the excerpt of the KEGG pathway in Figure 6.
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</b></p>
 +
<br>
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<center><img src="https://static.igem.org/mediawiki/2013/b/b8/ColourKegg.png" width="900" height="501"/></center>
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<p id="footer"><b>Figure 6: Excerpt of the simplified version of fatty acid elongation pathway. Coloured boxes and their metabolites correspond to the metabolites as indicated in Figure 5. 
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</b></p>
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<p>Next week most of the team is off to YSB 1.0 in London. We’re really looking forward to meeting the other UK iGEM teams!</p>
 
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<p> <center><img src="https://static.igem.org/mediawiki/2013/c/c2/Openday1.jpg"  /></center> </p>
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<p> These results further emphasise that although we created a model based on uncertain parameters, by embracing this uncertainty we have been able to make a model that gives us useful information – and that allows us to specify for every single prediction how certain we can be of getting it right, in particular towards the end of the pathway. </p>
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</div>
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<p> Similar data analysis was carried out on the rates of the reactions, shown in Figure 7. We focused on the reactions we had labelled AAT at the end of our pathway. These are thioesterase reactions directly responsible for the formation of palmitic and stearic acid. We can see that the rates for these reactions also fall within a relatively small range. </p>
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</li>
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<center><img src="https://static.igem.org/mediawiki/2013/e/ea/RateBoxPlot.jpg" width="497" height="432"/></center>
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            <li id="two"><a href="" onclick="blocking('text5'); return false;">Week 6</a>
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<p id="footer"><b>Figure 7: Reaction rates of key reactions in the fatty acid synthesis pathway.
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    <div id="text5">
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</b></p>
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                              <p> This has been a good week of collaborations for us! At the start of the week we had a Google hangout with Perdue to discuss their idea to standardise entry of parts into the registry. It emerged that there are many issues raised when it comes to standardising modelling. We agreed to help them with this. By the end of the week, this had somewhat grown...</p>
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          </div>
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<p>On Thursday we headed off to London for the very first Young Synthetic Biologists (YSB) conference. This was a big meeting of all the UK iGEM teams. On the first day we listened to presentations from each team in the morning. The afternoon featured workshops covering topics such as public engagement, business startups and bioart. </p>
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<div class="text3">
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<p> <b> <u> Conclusion </p> </b> </u>
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<p>The second day brought exciting news to the team. After our presentation, we had a lot of interest in our model. This led to a proposed collaboration with several other UK teams. Our aim is to each make a video tutorial on how to use various different software commonly used to model in iGEM, and to then make these videos accessible to future teams. Hopefully having introductions like this will encourage and inspire more people to embrace modelling in their projects!</p>
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<p> Kinetic Pathway modelling demands abundant information of the kinetic parameters. Literature research, however, showed that these were not available sufficiently or involved measurement errors. Hence this knowledge of parameter values often is uncertain. Therefore, we had to choose an approach that is able to deal with these limitations. Uncertainty modelling proved to be the most promising and useful tool for this. Even though the available data was limited, we managed to create a functioning kinetic model of the fatty acid synthesis pathway. This has not been done before and would not have been possible with any traditional approach. </p>
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<p> <center><img src="https://static.igem.org/mediawiki/2013/a/a0/Ysb12.jpg"  /></center> </p>
+
<p> A prime example of how our metabolic modelling work directly informed our experimental work is in our decision to biobrick the FabA gene (encoding β-hydroxydecanoyl-ACP dehydrase, shown by the DH_OH reactions in this model). Our uncertainty model had shown us that we would need more kinetic data on key enzymes. The least characterised reaction was catalyzed by the product of the fabA gene, therefore we wished to not only biobrick this gene, but a His-tag to purify the enzyme in order to experimental gauge its activity. </p>
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</div>
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<p> However, having taken pains to ensure our model was as realistic as possible, the idea of the insertion of a his-tag that could affect the activity of the enzyme seemed at odds to our overall goal. Therefore, we used further modelling technique to ensure the addition of this his tag would have as little overall bearing on the activity of the enzyme as possible. This can be found <a href="https://2013.igem.org/Team:Manchester/FabProteinModel" target="_blank">here</a><br>
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</li>
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        <li id="three"><a href="" onclick="blocking('text6'); return false;">Week 7</a>
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    <div id="text6">
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<p>This week’s been a quiet one, but it’s given some fairly substantial progress for the experimental and human practice teams. </p>
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<p>Experimentally, the FAS module arrived from Prof Mattheos Koffas of the Rensselaer Polytechnic Institute, Troy, New York. We successfully extracted lots of it (78ng/µl!). This FAS module will help increase fatty acid synthesis in our <i>E. coli</i> so we get a greater yield of product. We are incredibly grateful for Prof Koffas for his generosity in donating this module, as this will be a big help to our project. </p>
 
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<p>In human practice news, the team first had a meeting with James Leigh, a chemistry PhD student who has worked with the Oxbridge Biotech Roundtable. He gave the team some advice about how to organise a roundtable for a possible talk with industry we have in mind. It sounds like a lot of work, so we’re not sure if this is going to be a viable idea in the timescale we have left. There was also a meeting with Dr Catherine Rhodes, an expert in science ethics. She made us consider the rights needed to keep the Malaysian and Indonesian economies stable, and possible patenting issues with our project.<p>
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          </div>
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<p>Even more economic progress was made (having Matt on the team was definitely a good decision!). We got concrete figures showing there is a link between palm oil growth and deforestation, that there is going to be a sharp rise in the price of naturally grown palm oil in the future.</p>
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<div class="text3">
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<p> <b> <u> Future Applications: Potentials and Limitations </b> </u> </p>
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<p> We believe that this approach to modelling could have a big impact in terms of how Synthetic Biology is modelled in the future and demonstrates a method in which, by facing the uncertainty of modelling head-on and incorporating this into our approach in a principled manner, it is possible to produce valuable models. This is particularly important in the field of Synthetic Biology, where systems, even if well characterised in one organism, are unlikely to have the same parameters when expressed in another organism. </p>
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<p> <center><img src="https://static.igem.org/mediawiki/2013/8/85/GelExtractManc.jpg"  /></center> </p>
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<p> This approach gives us the ability to model complex and poorly experimentally measured systems, where previous attempts may have produced unrepresentative models. Since the Km values can be sampled from a distribution, the model can be used to determine outcomes that may not be obvious with the use of a single Km value. </p>
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</div>
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</li>
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        <li id="four"><a href="" onclick="blocking('text7'); return false;">Week 8</a>
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    <div id="text7">
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                              <p>This week finally brought good news in sponsorship! We would like to thank the Hain Daniels Group and Eccelso for their very generous donations to our team! They are very much appreciated and will no doubt help us travel to the European jamboree. </p>
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<p>The experimental team had some frustrations this week (it wouldn’t be real science without them!). After waiting a long time for our primers to arrive, they had some very confusing contamination after our PCR. The next challenge is to work out where it came from! The economics team drew up a list of things that need doing by time the project ends, and there’s lots of work to do! This week they found out that ‘sustainable’ palm oil isn’t as sustainable as you’d think. You can read much more about this on our ethics pages</p>
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<p> However, it is important to note that this method of modelling may not be appropriate in every case. The largest limitation of our use of this method is the inability of some of our simulations to reach steady state. This is likely to be a result of the random combination of parameter values. As the models were not fine-tuned, they will not always work. Although, we consider this as a potential strength as we can clearly highlight possible break points in the system that require further analysis. We show this in our own studies of β-hydroxydecanoyl-ACP dehydrase, described above. </p>
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<p>At the end of the week we had another iGEM social where we tried the Icelandic delicacy of Hákarl (fermented shark). As you can imagine, it tasted bad but smelled even worse! The vegetable chilli that Jess made the team was much more appetising...</p>
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<p> Synthetic Biology operates at the cutting edge of current knowledge. Therefore, it will unavoidably face the challenge of uncertainty. Building models with incorporated acknowledgment of uncertainty will yield model predictions with specified confidence intervals, and thus will lead to more robust design strategies for a vast range of engineered cellular machines. </p>
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</b></p>
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          </div>
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<p> <center><img src="https://static.igem.org/mediawiki/2013/a/a8/Hakarl.jpg" /></center> </p>
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<div class="text3">
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</div>
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<p> <b> <u> Appendices </p> </u> </b>
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</li>
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  </ul>
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  </li>
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<li id="month3"><a href="" onclick="blocking('submenu3'); return false;"><span><img src="https://static.igem.org/mediawiki/2013/e/ee/DownArrow2.png" width="30" height="30" /></span>August</a>
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<p> Here you can download all of the spreadsheets used in the creation of this model: </p>
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    <ul id="submenu3">      
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<p> The spreadsheets generated from our script can be found here:<br>
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        <li id="one"><a href="" onclick="blocking('text8'); return false;">Week 9</a>
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<a href="https://static.igem.org/mediawiki/2013/e/ef/Rates.pdf" target="_blank">RATES</a><br>
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<div id="text8">
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<a href="https://static.igem.org/mediawiki/2013/a/ac/Species.pdf">SPECIES</a><br>
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                              <p>This week was once again plagued with failed PCR runs, which then stalled the FadD knockout. It seems that the parameters on the thermocycler had been adjusted, leading to inconsistent results. Whilst we waited around for PCR cycles to complete, we decided to use the time to make up more stock solutions and media that we will (hopefully!) need in the near-future. That and to make ourselves feel a little more productive...</p>
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<br>
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<b>Nomenclature of main metabolites</b><br>
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<img src="https://static.igem.org/mediawiki/2013/8/84/BlueTable1.png" width="295" height="569"/><br>
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<img src="https://static.igem.org/mediawiki/2013/d/d5/BlueTable2.png" width="294" height="333"/><br>
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<br>
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<img src="https://static.igem.org/mediawiki/2013/9/9d/Greentable1.png" width="261" height="384"/><br>
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<img src="https://static.igem.org/mediawiki/2013/d/d1/Greentable2.png" width="261" height="143"/><br>
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<p>The ethics research is still coming along nicely. This week the team did more research into what policies are currently in place to protect rainforests, and looked into several case studies detailing previous instances where a synthetic alternative to a naturally occurring product has been introduced, amongst other things. We also contacted a number of companies in the hopes that one or two of them will be interested in sharing their opinions on the palm oil industry with us.</p>
 
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<p>We also of course had more socials! First up was a Thai meal that lead onto a Gypsy jazz gig at a small bar in town, which the team thought was great. Then on friday we spent the evening chatting on the roof terrace of Marco’s flat, and took some really blurry photos (as you can see below)!</p>
 
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<p> <center><img src="https://static.igem.org/mediawiki/2013/3/3e/Marcosflat.jpg"  /></center> </p>
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          </div>
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</div>
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</li>
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            <li id="two"><a href="" onclick="blocking('text9'); return false;">Week 10</a>
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    <div id="text9">
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                              <p>It’s Week 10 already? Not sure how that happened. The pressure is on (...even more) now! The FadD knockout is still proving elusive, we’re sort of losing hope that it will ever work. This week will be our final attempt to complete it. Fingers crossed!</p>
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<p>This week we had a meeting with Eriko and Rainer to discuss our progress with the project. They gave us some invaluable advice regarding the finer details of our experimental protocols, so we will put them into practice soon and hope to see some results! We received approval to order our 2 required genes: 𝛥9 and 𝛥12. This means that we should have them with us by the end of August. It will be a push to get them ready in time for the BioBrick deadline, but we’re hopeful that we will make it.<br>
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          </div>        
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We have exciting news regarding our future fatty acid analyses. We found a new supervisor in Dr. Nik Rattray, a postdoc working with Prof. Roy Goodacre. Nik works with Orbitrap LC-MS, and will be helping us to characterise the fatty acid profiles of our constructs (once we make them!).<br>
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Next week we will be cloning the FabA gene out of the <i>E. coli</i> BL21 (DE3) genome, which we extracted from wild-type this week. The primers needed for the cloning of FabA were also ordered.</p>
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<p>The economics and ethics research is feeling close to completion, and the write up has begun! Now it will be a race to get it all on the wiki looking pretty in time. No small feat, but the ethics guys can do it!.</p>
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</div>  
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<div class="leftbar">
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<p>The project is getting increasingly stressful, which is seeing a rise in our team socialising time! Coincidence? I think not. This week the team visited Tim’s place for food on Saturday, then went to a few bars in town, our favourite of which was The Alchemist. If they serve drinks in conical flasks it still counts as research, right?</p>
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<p> <center><img src="https://static.igem.org/mediawiki/2013/4/40/Alchemistcrew.jpg"  /></center> </p>
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</div>
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        <li id="three"><a href="" onclick="blocking('text10'); return false;">Week 11</a>
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    <div id="text10">
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                              <p>Sorry, this may be a long one! We started out the week with a big team meeting, during which we came up with a list of everything that needs to be done this week. Busy would be an understatement. We even had to seek out permission to enter the building at 7am, a first for this project! <br>
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The work was split into 4 sections: 1. Cloning and inserting ribosomal binding site (RBS) gene into pUC18 vector, 2. Cloning of FabA from WT <i>E. coli</i> BL21 (DE3), 3. FadD knockout, 4. LC-MS characterisation.</p>
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<p>1. RBS BioBricks from the kit were ligated into plasmid, transformed into <i>E. coli</i> DH5-alpha (?), and miniprepped. (this is probably wrong. Rob or Tim clarify please)</p>
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<p>2. We really want to BioBrick FabA because it is an important part of the fatty acid biosynthesis pathway of <i>E. coli</i> that is yet to be submitted to the Registry, and also we would really like to attempt to measure the kinetic properties of the enzyme to improve our model. This week we cloned the gene from <i>E. coli</i> BL21 (DE3) using PCR and stored in the freezer. The team are now thinking on the best ways to characterise this gene.</p>
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<p>3. Sadly, this week we had to say goodbye to our hopes of achieving knockout of the FadD gene. 10 long weeks and nothing to show for it (other than a significant improvement in our PCR and gel electrophoresis skills)! </p>
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<p>4. We met with Nik who guided us through the quenching and metabolite extraction of prokaryotic cells. Initially we are running reference samples through Orbitrap LC-MS (WT <i>E. coli</i> BL21 (DE3) cells in different media, solid and liquid fractions of authentic palm oil), which we will then compare to the fatty acid profiles of our constructs. Nik also kept us entertained with stories about how the Orbitrap technology was developed in a cellar here in Manchester, and showed us a signed mass spectrometer by Alexander Makarov himself!</p>
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<p>This week’s social saw us at Jess’ house once more, this time for delicious Malaysian food and a film night after a particularly gruelling day in the lab. It goes without saying that everyone enjoyed letting their hair down for a bit.</p>
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<p> <center><img src="https://static.igem.org/mediawiki/2013/1/1b/Rsz_board.jpg"  /></center> </p>
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        <li id="four"><a href="" onclick="blocking('text11'); return false;">Week 12</a>
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    <div id="text11">
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                              <p>Disaster struck this week! The stock of DH5-a cells from next door’s lab became contaminated with a mystery ampicillin resistant plasmid, meaning our fabA transformed cells are useless. After a week of success, it was very disappointing to find this out. But hey, these things can happen in science. Time to put this behind us, find some new cells and start again! A Skype conversation with our supervisor was very reassuring, and we’re confident we can easily pull this back.</p>
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<p> Meanwhile, in the ethics department, the research is done and the write-up for the wiki has started! We’ve found so much we want to write about, our main issue is wondering how we’re going to do it! It’s a good job we didn’t leave this until a week before wikifreeze…</p>
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<p>Monday also saw Divita leave to go on holiday, so Tan (well, her sister!) cooked the team a great meal to say goodbye.</p>
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<p> <center><img src="https://static.igem.org/mediawiki/2013/8/8c/Piclab5.jpg"  /></center> </p>
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          <li id="month4"><a href="" onclick="blocking('submenu4'); return false;"><span><img src="https://static.igem.org/mediawiki/2013/e/ee/DownArrow2.png" width="30" height="30" /></span>September</a>
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        <li id="one"><a href="" onclick="blocking('text12'); return false;">Week 13</a>
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<div id="text12">
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                              <p>This week has flown by! Partly because of how busy we were, but probably because it was a bank holiday on Monday so we’ve had a 4 day week (got to adhere to health and safety regulations!)</p>
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<p>Tuesday finally saw the arrival of our synthesised delta 9 and delta 12 genes, so the main objective of our project can finally begin!. Without any hesitation we hydrated them, transformed them into <i>E. coli </i>, and miniprepped them to get a lovely stock of DNA ready for us to use for a biobrick! We also inserted fabA in to a blunt-end vector and transformed it to get more DNA to work with. Next, they were all digested ready to ligate into the iGEM submission vector on Friday. Everything was going well right up to our awful gel extraction yields, meaning biobricks didn’t happen this week! It was very disappointing, but our weekend will be spent finding ways to improve our techniques.</p>
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<p> Once again this week we said goodbye to certain members of the team. Not one, not two, but THREE team members left us this week (obviously optimistic we’d have the project finished by now!). Now that Tim, Marco and Elsa are gone, we have 3 on the lab team and 2 on the modelling team.  With not long to go until the biobrick submission deadline, here’s hoping we work well under pressure! </p>
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                              <p>This week was filled with frustration, as our gel extractions refused to cooperate with us! Luckily however, Lorna soon became a gel extraction pro, and our DNA yield soon increased to workable levels. </p>
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<p>This week we also ordered our polo shirts, and we got the (correct!) sequencing results back from our fabA gene. </p>
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                              <p>It’s starting to feel like all of the effort we’ve been putting into the project will pay off! This week we managed to get our genes of interest (fabA, delta 9 and delta 12) into the iGEM submission plasmid! Sequencing showed that our genes are in fact ligated, and so we eagerly arranged a fedEX shipment to send our samples on their merry way to Boston. It’s not over though! We still needed to successfully ligate our genes into an expression plasmid. We chose pSB1C3 with a ribosomal binding site (RBS) and promoter (P) (Part BB1_ K608002). After a few seemingly failed attempts (our colonies were very small and took a while longer than expected to grow), we decided to test digest anyway because what did we have to lose (except all hope)? Excitingly however, the test digestions suggested that we’d put the genes into the expression plasmid! Time caught up with us and so we will be characterising next week. We think that the small colonies may be a result of the constitutive promoter used, and in an ideal world we’d check this theory. With one week to go though we will just have to leave it to any future iGEM teams out there!</p>
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<p>The pressure is very much on now, the whole team is feeling it. We’re desperately trying to juggle a chaotic lab schedule, writing up the wiki and making the presentation/poster for the jamboree. However there's still human practices to be done! In addition to all our lab work, we met with a group of local environmentalists and spoke to large industries to aid us in gauging what the public and industry really think about synthetic biology. </p>
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                              <p>This is it! The final stretch to wikifreeze and the pressure is hotting up. At the start of the week we inoculated our RBS/P and Delta9/Delta12/fabA constructs in FAS media containing the fatty acids we needed to feed the bacteria with. We left our samples in the very capable hands of Dr Rattray who ran our samples on the Orbitrap LC-MS so we could see if our fatty acid profile had changed. The results finally came through the day before wikifreeze, and we were thrilled to find out  that our biobricks had worked! </p>
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<p>There’s no time to celebrate though. It’s the day of wiki freeze and we’re furiously working to get our wiki finished. Talk about cutting it fine!</p>
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Latest revision as of 21:32, 28 October 2013

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Safety

Summary

Working with a pathway as large and uncharacterised as the fatty acid biosynthesis presented many challenges, the most important of which was the lack of reliable, experimentally established kinetic values for many of the key reactions. Our solution was to create a model that explicitly acknowledges this lack of data and the resulting uncertainty, using Monte Carlo sampling from plausible parameter value distributions -- enabling us to produce model predictions with confidence intervals. We believe that this unusual and innovative modelling strategy can potentially serve as a general principled approach to handling parameter uncertainty in the future. Synthetic Biology will always operate at the cutting edge of current knowledge and thus will unavoidably face the challenge of uncertainty. Building models with incorporated acknowledgment of uncertainty will yield model predictions with specified confidence intervals, and thus will lead to more robust design strategies for a wide range of engineered cellular machines.

Aim

To use uncertainty modelling to model E. coli fatty acid biosynthesis.

Early modelling attempts using traditional methods of modelling were largely unsuccessful, due to the the nature of the fatty acid biosynthesis pathway, and the lack of experimentally defined kinetic values. Rather than use models that were arbitrary or lacked information, we decided to use a less traditional method, based on Monte Carlo sampling, that can give us a clear idea of what the uncertainty of our predictions might be. By embracing this uncertainty, we hoped to create a model with practical, representative results.

Objectives

  • To build the first kinetic model of fatty acid biosynthesis in E. coli using uncertainty modeling
  • To represent the fatty acid production in our system: E.c(oil)i
  • To identify areas of the pathway requiring further study in the lab
  • To develop a method of modelling that can be used in the absence of a fully characterised pathway

Introduction

Fatty acid biosynthesis is a process that occurs in all living organisms. Glucose is converted into acetyl-CoA through the citric acid cycle, which is fed into the fatty acid biosynthesis pathway. Here it reacts with malonyl-CoA to form a four carbon compound. The four carbon compound is then reduced and dehydrated via four successive steps, executed, with the help of NADPH, by the enzymes as indicated in Figure 1. To this resulting C4 body, another malonyl-CoA reacts to form a C6 body - which is converted the same manner as the previous C4 body. A number of unchanging enzymes act on the intermediates of this cyclic pathway to ultimately produce fatty acids. From the initial reaction to the end products the whole pathway numbers 43 reactions, about 60 metabolites and 267 parameters.

In synthetic biology two main classes of computational models are commonly used: constraint-based genome-scale models and differential-equation-based dynamic models. In our project, we were interested in the concentrations of compounds and their dynamic changes as well as the reactions with the highest control over the fatty acid synthesis pathway. As our analysis would not be possible with a purely constraint-based model, we chose to use a dynamic model. However, to use a dynamic model one needs to know the enzyme kinetic parameters, and these are often unknown or very unreliable for enzymes. Uncertainty can be due to:

  • experimental uncertainty
  • in vitro measurements of enzyme kinetics are not always representative of in vivo conditions
  • compound concentrations often have dynamic changes
  • We wanted to account for the uncertainty in the fatty acid synthesis pathway parameter data by using a new “uncertainty modelling” approach, which can potentially serve as a principled approach to handling parameter uncertainty in the future.

    Building models with incorporated acknowledgment of uncertainty will produce specified confidence intervals for all model predictions and thus could lead to robust design of engineered cellular machines of fatty acid synthesis and beyond.

    Method

  • Collected parameter values from literature
  • Using the online database BRENDA, we searched for published parameter values for every enzyme in the pathway. In our search we discovered that the published data on the value of parameters in the E. coli fatty acid biosynthesis pathway is limited. Hence, we decided we needed to take this into account in our model.

  • Categorized parameters into three groups
  • Group 1: ☑Mean ☑Standard Deviation
    Group 2: ☑Mean ☐Standard Deviation
    Group 3: ☐Mean ☐Standard Deviation

  • Filling in the missing information for each parameter
  • In the case of group one, both the mean and standard deviation were collected from the literature and used to determine the probability distribution. In the case of group two, we used the mean and standard deviation of all enzymes of the same class or subclass with known kinetic parameters. In the case of group three, we used the standard deviation obtained from all enzymes of the same sub-class to create a distribution. Here is a file with the distributions of enzyme classes and subclasses. Below is a spreadsheet of each parameter value. The source of each parameter value is hyperlinked in blue.



  • Random sampling values from each parameter distribution
  • Once each parameter had a probability distribution associated with it, we randomly sampled values from each parameter distribution to run our model simulation. This was done by constructing an initial model in Copasi, using appropriate enzyme kinetic equations. The rate equations used in our model can be generalised as follows:



    We used these rate equations to complete our model of the fatty acid synthesis pathway by adding the thioesterase reactions required for the production of the final fatty acids as well as the reactions catalyzed by FadD. Once our model was complete enough to represent our system we exported this from COPASI in SBML format and converted it to a PySCeS compatible file. PySCeS uses a set of non-linear differential equations are used to obtain both structural and kinetic information about the system from these randomly generated kinetic values. Below is pseudocode of our workflow, the full script to randomly sample the parameters and generate model predictions can be downloaded here.



  • Creation of 1,000 models with distribution of predictions
  • We automated the generation of models to create a collection of 1,000 models. From there we were able to determine the uncertainty in our model predictions: instead of a single prediction, we have a distribution of predictions from a large collection of plausible models.

    Results


    The concentrations of the metabolites was outputted in tables, as depicted in Figure 3. Each line represents one simulation with ten different time points within 100 seconds. The whole data set of all simulations was then attributed with colours according concentration values. Another table was generated out of this chart according to the ordinal data obtained from colouring the metabolite concentrations. This was done to further improve ease of work and making the data more visual. Figure 4 shows the summary of this qualitative concentration distribution for each metabolite. Again, the brighter green a cell is in colour, the more often simulations rendered metabolite concentrations in the specified concentration interval. For example, the last metabolite in the table C18CoA is bright green, because all 41 simulations rendered between 0.01 - 1 mM. Out of this table, a clear distribution becomes obvious: Except for the first six initial replenishing reactions, all metabolite concentrations are within a small reasonable range mostly between 0.01 - 1 mM. Interestingly, in the reaction towards the end of the pathway, which are responsible for removing the metabolites from the system and therefore give rise to stearic and palmitic acid (our desired products) the range of results appears to be significantly narrower, despite the uncertainty.

    The analysis of the data shows clearly, that due to a small and reasonable range of metabolite concentrations which stabilises towards the end of the model, a high validity of our functioning model can be safely assumed and demonstrates that the uncertainty is not globally deleterious. Even though the model was working with high uncertainties in data, the output is always within a valid range.

    Upon analysing the degree of certainty in our model, and finding that it was at a level that we believe is suitable for further analysis, we were able to create a series of boxplots showing the range of values found within our simulations for species accumulation after 100 seconds. We focused on the longer chain fatty acids, which are the engineering target of our pathway. The order in which the species are shown in the box plots, Figure 5, is also the order in which they are formed. This is also shown in Figure 6, where the colour corresponds to the colour of the bar on the box plot.


    These results further emphasise that although we created a model based on uncertain parameters, by embracing this uncertainty we have been able to make a model that gives us useful information – and that allows us to specify for every single prediction how certain we can be of getting it right, in particular towards the end of the pathway.

    Similar data analysis was carried out on the rates of the reactions, shown in Figure 7. We focused on the reactions we had labelled AAT at the end of our pathway. These are thioesterase reactions directly responsible for the formation of palmitic and stearic acid. We can see that the rates for these reactions also fall within a relatively small range.

    Conclusion

    Kinetic Pathway modelling demands abundant information of the kinetic parameters. Literature research, however, showed that these were not available sufficiently or involved measurement errors. Hence this knowledge of parameter values often is uncertain. Therefore, we had to choose an approach that is able to deal with these limitations. Uncertainty modelling proved to be the most promising and useful tool for this. Even though the available data was limited, we managed to create a functioning kinetic model of the fatty acid synthesis pathway. This has not been done before and would not have been possible with any traditional approach.

    A prime example of how our metabolic modelling work directly informed our experimental work is in our decision to biobrick the FabA gene (encoding β-hydroxydecanoyl-ACP dehydrase, shown by the DH_OH reactions in this model). Our uncertainty model had shown us that we would need more kinetic data on key enzymes. The least characterised reaction was catalyzed by the product of the fabA gene, therefore we wished to not only biobrick this gene, but a His-tag to purify the enzyme in order to experimental gauge its activity.

    However, having taken pains to ensure our model was as realistic as possible, the idea of the insertion of a his-tag that could affect the activity of the enzyme seemed at odds to our overall goal. Therefore, we used further modelling technique to ensure the addition of this his tag would have as little overall bearing on the activity of the enzyme as possible. This can be found here

    Future Applications: Potentials and Limitations

    We believe that this approach to modelling could have a big impact in terms of how Synthetic Biology is modelled in the future and demonstrates a method in which, by facing the uncertainty of modelling head-on and incorporating this into our approach in a principled manner, it is possible to produce valuable models. This is particularly important in the field of Synthetic Biology, where systems, even if well characterised in one organism, are unlikely to have the same parameters when expressed in another organism.

    This approach gives us the ability to model complex and poorly experimentally measured systems, where previous attempts may have produced unrepresentative models. Since the Km values can be sampled from a distribution, the model can be used to determine outcomes that may not be obvious with the use of a single Km value.

    However, it is important to note that this method of modelling may not be appropriate in every case. The largest limitation of our use of this method is the inability of some of our simulations to reach steady state. This is likely to be a result of the random combination of parameter values. As the models were not fine-tuned, they will not always work. Although, we consider this as a potential strength as we can clearly highlight possible break points in the system that require further analysis. We show this in our own studies of β-hydroxydecanoyl-ACP dehydrase, described above.

    Synthetic Biology operates at the cutting edge of current knowledge. Therefore, it will unavoidably face the challenge of uncertainty. Building models with incorporated acknowledgment of uncertainty will yield model predictions with specified confidence intervals, and thus will lead to more robust design strategies for a vast range of engineered cellular machines.

    Appendices

    Here you can download all of the spreadsheets used in the creation of this model:

    The spreadsheets generated from our script can be found here:
    RATES
    SPECIES

    Nomenclature of main metabolites