Team:CSU Fort Collins/HVP/

From 2014.igem.org

(Difference between revisions)
m (Created page with "{{CSS/Main}} <html> <!--main content --> <link href='http://fonts.googleapis.com/css?family=Bitter:400,700,400italic' rel='stylesheet' type='text/css'> <link href='http://font...")
Line 1: Line 1:
{{CSS/Main}}
{{CSS/Main}}
-
 
<html>
<html>
-
<!--main content -->
+
 
 +
 
 +
<title>High-Value Product</title>
 +
 
 +
<head>
<link href='http://fonts.googleapis.com/css?family=Bitter:400,700,400italic' rel='stylesheet' type='text/css'>
<link href='http://fonts.googleapis.com/css?family=Bitter:400,700,400italic' rel='stylesheet' type='text/css'>
<link href='http://fonts.googleapis.com/css?family=Muli:300,400' rel='stylesheet' type='text/css'>
<link href='http://fonts.googleapis.com/css?family=Muli:300,400' rel='stylesheet' type='text/css'>
-
 
-
<body style="background-size:100%; background-color:#EFEFEF">
 
<style type="text/css">
<style type="text/css">
 +
/* http://meyerweb.com/eric/tools/css/reset/
 +
  v2.0 | 20110126
 +
  License: none (public domain)
 +
*/
 +
 +
html, body, div, span, applet, object, iframe, p, blockquote, pre,
 +
a, abbr, acronym, address, big, cite, code,
 +
del, dfn, em, img, ins, kbd, q, s, samp,
 +
small, strike, strong, sub, sup, tt, var,
 +
b, u, i, center,
 +
dl, dt, dd, ol, ul, li,
 +
fieldset, form, label, legend,
 +
table, caption, tbody, tfoot, thead, tr, th, td,
 +
article, aside, canvas, details, embed,
 +
figure, figcaption, footer, header, hgroup,
 +
menu, nav, output, ruby, section, summary,
 +
time, mark, audio, video {
 +
  margin: 0;
 +
  padding: 0;
 +
  border: 0;
 +
  font-size: 90%;
 +
  font: inherit;
 +
  vertical-align: baseline;
 +
}
 +
/* HTML5 display-role reset for older browsers */
 +
article, aside, details, figcaption, figure,
 +
footer, header, hgroup, menu, nav, section {
 +
  display: block;
 +
}
 +
body {
 +
  line-height: 1;
 +
}
 +
ol, ul {
 +
  list-style: none;
 +
}
 +
blockquote, q {
 +
  quotes: none;
 +
}
 +
blockquote:before, blockquote:after,
 +
q:before, q:after {
 +
  content: '';
 +
  content: none;
 +
}
 +
table {
 +
  border-collapse: collapse;
 +
  border-spacing: 0;
 +
}
 +
 +
#menubar > ul{
 +
  vertical-align:center;
 +
  font-size:14px;
 +
  font-family:'Times New Roman',serif;
 +
}
 +
.firstHeading{
.firstHeading{
-
    display:none;
+
display:none;
}
}
-
A{
+
 
-
    color:#222222;
+
#textbox span{
 +
  color:#E6B800;
}
}
-
A:visited{
+
.photo{
-
    color:#222222;
+
  width:70%;
}
}
-
A:hover{
+
table{
-
    opacity:0.3;
+
  font-family:'Muli',sans-serif;
-
    text-decoration:none;
+
}
}
-
h1,h2,h3,h4,h5,h6{
+
body{
-
    font-family:'Bitter', serif;
+
  background-color:#EFEFEF;
-
    text-decoration:none;
+
  line-height:1.3;
-
    font-weight:700;
+
  font-size:90%;
-
    color:#222222;
+
-
    border:none;
+
}
}
-
.button{
+
#cssmenu {
-
    margin-left:15px;
+
  width:100%;
-
    margin-right:15px;
+
  height: 44px;
-
    padding:10px;
+
  background: #222222;
-
    font-family:'Bitter', serif;
+
  position:relative;
-
    font-size:14px;
+
  text-align:center;
-
    width:5em;
+
  margin:auto;
}
}
-
.sbutton{
+
#cssmenu ul {
-
    padding:5px 0px;
+
  list-style: none;
-
    font-family:'Bitter', serif;
+
  padding: 0;
-
    font-size:14px;
+
  margin: 0;
 +
  line-height: 1;
}
}
-
nav ul{
+
#cssmenu > ul {
-
    position:relative;
+
  position: relative;
-
    list-style:none;
+
  display: block;
-
    white-space:nowrap;
+
  background: #222222;
 +
  width: 100%;
 +
  z-index: 500;
}
}
-
nav ul li{
+
#cssmenu:after,
-
    display:inline-block;
+
#cssmenu > ul:after {
-
    white-space:nowrap;
+
  content: '.';
 +
  display: block;
 +
  clear: both;
 +
  visibility: hidden;
 +
  line-height: 0;
 +
  height: 0;
}
}
-
nav ul ul {
+
#cssmenu.align-right > ul > li {
-
    display: none;
+
  float: right;
-
    position:absolute;
+
-
    margin-left: 0;
+
-
    margin-top: 10px;
+
}
}
-
nav ul ul li{
+
#cssmenu.align-center ul {
-
    display: block;
+
  text-align: center;
-
    text-align: left;
+
-
    float: none;
+
}
}
-
nav ul ul .button {
+
#cssmenu.align-center ul ul {
-
    margin-left: 0;
+
  text-align: left;
-
    padding-left: 0;
+
}
}
-
nav ul li a{
+
#cssmenu > ul > li {
-
    display:block !important;
+
  display: inline-block;
 +
  position: relative;
 +
  margin: 0;
 +
  padding: 0;
 +
}
 +
#cssmenu > ul > #menu-button {
 +
  display: none;
 +
}
 +
#cssmenu ul li a {
 +
  display: block;
 +
  font-family: Helvetica, sans-serif;
 +
  text-decoration: none;
 +
}
 +
#cssmenu > ul > li > a {
 +
  font-size: 14px;
 +
  font-weight: bold;
 +
  padding: 15px 20px;
 +
  color: #efefef;
 +
  text-transform: uppercase;
 +
  -webkit-transition: color 0.25s ease-out;
 +
  -moz-transition: color 0.25s ease-out;
 +
  -ms-transition: color 0.25s ease-out;
 +
  -o-transition: color 0.25s ease-out;
 +
  transition: color 0.25s ease-out;
 +
}
 +
#cssmenu > ul > li.has-sub > a {
 +
  padding-right: 32px;
 +
}
 +
#cssmenu > ul > li:hover > a {
 +
  color: #E6B800;
 +
}
 +
#cssmenu li.has-sub::after {
 +
  display: block;
 +
  content: '';
 +
  position: absolute;
 +
  width: 0;
 +
  height: 0;
 +
}
 +
#cssmenu > ul > li.has-sub::after {
 +
  right: 10px;
 +
  top: 20px;
 +
  border: 5px solid transparent;
 +
  border-top-color: #efefef;
 +
}
 +
#cssmenu > ul > li:hover::after {
 +
  border-top-color: #E6B800;
 +
}
 +
#indicatorContainer {
 +
  position: absolute;
 +
  height: 12px;
 +
  width: 100%;
 +
  bottom: 0px;
 +
  overflow: hidden;
 +
  z-index: -1;
 +
}
 +
#pIndicator {
 +
  position: absolute;
 +
  height: 0;
 +
  width: 100%;
 +
  border: 12px solid transparent;
 +
  border-top-color: #2b2f3a;
 +
  z-index: -2;
 +
  -webkit-transition: left .25s ease;
 +
  -moz-transition: left .25s ease;
 +
  -ms-transition: left .25s ease;
 +
  -o-transition: left .25s ease;
 +
  transition: left .25s ease;
 +
}
 +
#cIndicator {
 +
  position: absolute;
 +
  height: 0;
 +
  width: 100%;
 +
  border: 12px solid transparent;
 +
  border-top-color: #2b2f3a;
 +
  top: -12px;
 +
  right: 100%;
 +
  z-index: -2;
 +
}
 +
#cssmenu ul ul {
 +
  position: absolute;
 +
  left: -9999px;
 +
  top: 70px;
 +
  opacity: 0;
 +
  -webkit-transition: opacity .3s ease, top .25s ease;
 +
  -moz-transition: opacity .3s ease, top .25s ease;
 +
  -ms-transition: opacity .3s ease, top .25s ease;
 +
  -o-transition: opacity .3s ease, top .25s ease;
 +
  transition: opacity .3s ease, top .25s ease;
 +
  z-index: 1000;
 +
}
 +
#cssmenu ul ul ul {
 +
  top: 37px;
 +
  padding-left: 5px;
 +
}
 +
#cssmenu ul ul li {
 +
  position: relative;
 +
}
 +
#cssmenu > ul > li:hover > ul {
 +
  left: auto;
 +
  top: 44px;
 +
  opacity: 1;
 +
}
 +
#cssmenu.align-right > ul > li:hover > ul {
 +
  left: auto;
 +
  right: 0;
 +
  opacity: 1;
 +
}
 +
#cssmenu ul ul li:hover > ul {
 +
  left: 170px;
 +
  top: 0;
 +
  opacity: 1;
 +
}
 +
#cssmenu.align-right ul ul li:hover > ul {
 +
  left: auto;
 +
  right: 170px;
 +
  top: 0;
 +
  opacity: 1;
 +
  padding-right: 5px;
 +
}
 +
#cssmenu ul ul li a {
 +
  width: 130px;
 +
  border-bottom: 1px solid #eeeeee;
 +
  padding: 10px 20px;
 +
  font-size: 12px;
 +
  color: #222222;
 +
  background: white;
 +
  -webkit-transition: all .35s ease;
 +
  -moz-transition: all .35s ease;
 +
  -ms-transition: all .35s ease;
 +
  -o-transition: all .35s ease;
 +
  transition: all .35s ease;
 +
}
 +
#cssmenu.align-right ul ul li a {
 +
  text-align: right;
 +
}
 +
#cssmenu ul ul li:hover > a {
 +
  background: white;
 +
  color: #E6B800;
 +
}
 +
#cssmenu ul ul li:last-child > a,
 +
#cssmenu ul ul li.last > a {
 +
  border-bottom: 0;
 +
}
 +
#cssmenu > ul > li > ul::after {
 +
  content: '';
 +
  border: 6px solid transparent;
 +
  width: 0;
 +
  height: 0;
 +
  border-bottom-color: #efefef;
 +
  position: absolute;
 +
  top: -12px;
 +
  left: 30px;
 +
}
 +
#cssmenu.align-right > ul > li > ul::after {
 +
  left: auto;
 +
  right: 30px;
 +
}
 +
#cssmenu ul ul li.has-sub::after {
 +
  border: 4px solid transparent;
 +
  border-left-color: #222222;
 +
  right: 10px;
 +
  top: 12px;
 +
  -moz-transition: all .2s ease;
 +
  -ms-transition: all .2s ease;
 +
  -o-transition: all .2s ease;
 +
  transition: all .2s ease;
 +
  -webkit-transition: -webkit-transform 0.2s ease, right 0.2s ease;
 +
}
 +
#cssmenu.align-right ul ul li.has-sub::after {
 +
  border-left-color: transparent;
 +
  border-right-color: #222222;
 +
  right: auto;
 +
  left: 10px;
 +
}
 +
#cssmenu ul ul li.has-sub:hover::after {
 +
  border-left-color: #efefef;
 +
  right: -5px;
 +
  -webkit-transform: rotateY(180deg);
 +
  -ms-transform: rotateY(180deg);
 +
  -moz-transform: rotateY(180deg);
 +
  -o-transform: rotateY(180deg);
 +
  transform: rotateY(180deg);
 +
}
 +
#cssmenu.align-right ul ul li.has-sub:hover::after {
 +
  border-right-color: #efefef;
 +
  border-left-color: transparent;
 +
  left: -5px;
 +
  -webkit-transform: rotateY(180deg);
 +
  -ms-transform: rotateY(180deg);
 +
  -moz-transform: rotateY(180deg);
 +
  -o-transform: rotateY(180deg);
 +
  transform: rotateY(180deg);
 +
}
 +
@media all and (max-width: 800px), only screen and (-webkit-min-device-pixel-ratio: 2) and (max-width: 1024px), only screen and (min--moz-device-pixel-ratio: 2) and (max-width: 1024px), only screen and (-o-min-device-pixel-ratio: 2/1) and (max-width: 1024px), only screen and (min-device-pixel-ratio: 2) and (max-width: 1024px), only screen and (min-resolution: 192dpi) and (max-width: 1024px), only screen and (min-resolution: 2dppx) and (max-width: 1024px) {
 +
  #cssmenu {
 +
    width: 100%;
 +
  }
 +
  #cssmenu.align-center ul {
 +
    text-align: left;
 +
  }
 +
  #cssmenu.align-right > ul > li {
 +
    float: none;
 +
  }
 +
  #cssmenu ul {
 +
    width: auto;
 +
  }
 +
  #cssmenu .submenuArrow,
 +
  #cssmenu #indicatorContainer {
 +
    display: none;
 +
  }
 +
  #cssmenu > ul {
 +
    height: auto;
 +
    display: block;
 +
    color:#EFEFEF;
 +
  }
 +
  #cssmenu > ul > li {
 +
    float: none;
 +
  }
 +
  #cssmenu li,
 +
  #cssmenu > ul > li {
 +
    display: none;
 +
    color:white;
 +
  }
 +
  #cssmenu ul ul,
 +
  #cssmenu ul ul ul,
 +
  #cssmenu ul > li:hover > ul,
 +
  #cssmenu ul ul > li:hover > ul,
 +
  #cssmenu.align-right ul ul,
 +
  #cssmenu.align-right ul ul ul,
 +
  #cssmenu.align-right ul > li:hover > ul,
 +
  #cssmenu.align-right ul ul > li:hover > ul {
 +
    position: relative;
 +
    left: auto;
 +
    top: auto;
 +
    opacity: 1;
 +
    padding-left: 0;
 +
    padding-right: 0;
 +
    right: auto;
 +
  }
 +
  #cssmenu ul .has-sub::after {
 +
    display: none;
 +
  }
 +
  #cssmenu ul li a {
 +
    padding: 12px 20px;
 +
  }
 +
  #cssmenu ul ul li a {
 +
    border: 0;
 +
    background: none;
 +
    width: auto;
 +
    padding: 8px 35px;
 +
    color:white;
 +
  }
 +
  #cssmenu.align-right ul ul li a {
 +
    text-align: left;
 +
  }
 +
  #cssmenu ul ul li:hover > a {
 +
    background:none;
 +
    color: #ec9359;
 +
  }
 +
  #cssmenu ul ul ul a {
 +
    padding: 8px 50px;
 +
  }
 +
  #cssmenu ul ul ul ul a {
 +
    padding: 8px 65px;
 +
  }
 +
  #cssmenu ul ul ul ul ul a {
 +
    padding: 8px 80px;
 +
  }
 +
  #cssmenu ul ul ul ul ul ul a {
 +
    padding: 8px 95px;
 +
  }
 +
  #cssmenu > ul > #menu-button {
 +
    display: block;
 +
    cursor: pointer;
 +
  }
 +
  #cssmenu #menu-button > a {
 +
    padding: 14px 20px;
 +
  }
 +
  #cssmenu ul.open li,
 +
  #cssmenu > ul.open > li {
 +
    display: block;
 +
  }
 +
  #cssmenu > ul.open > li#menu-button > a {
 +
    color: #fff;
 +
    border-bottom: 1px solid rgba(150, 150, 150, 0.1);
 +
  }
 +
  #cssmenu ul ul::after {
 +
    display: none;
 +
  }
 +
  #cssmenu #menu-button::after {
 +
    display: block;
 +
    content: '';
 +
    position: absolute;
 +
    height: 3px;
 +
    width: 22px;
 +
    border-top: 2px solid #efefef;
 +
    border-bottom: 2px solid #efefef;
 +
    right: 20px;
 +
    top: 15px;
 +
  }
 +
  #cssmenu #menu-button::before {
 +
    display: block;
 +
    content: '';
 +
    position: absolute;
 +
    height: 3px;
 +
    width: 22px;
 +
    border-top: 2px solid #efefef;
 +
    right: 20px;
 +
    top: 25px;
 +
  }
 +
  #cssmenu ul.open #menu-button::after,
 +
  #cssmenu ul.open #menu-button::before {
 +
    border-color: #fff;
 +
  }
 +
}
 +
.icon{
 +
  width:30px;
 +
}
 +
.wrapper2{
 +
  width:20%;
 +
  padding:20px 0px 20px 20px;
 +
  display:inline-block;
 +
  margin:auto;
 +
  vertical-align:top;
 +
}
 +
.wrapper{
 +
  width:90%;
 +
  padding:20px 0px 20px 20px;
 +
  padding-left:0;
 +
  margin:auto;
 +
  vertical-align: top;
 +
}
 +
.page, .container{
 +
  width:50%;
 +
  min-width:600px;
 +
  max-width:800px;
 +
  padding:30px;
 +
  margin:auto;
 +
  text-align:left;
 +
  font-family:'Muli',sans-serif;
 +
  display:inline-block;
 +
}
 +
.page a, .container a{
 +
  color:#222222;
 +
}
 +
.page a:hover, .container a:hover{
 +
  color:#E6B800;
 +
  text-decoration:none;
}
}
-
nav ul li:hover > ul {
+
h1{
-
     display:block;
+
  color:#222222;
 +
  font-family:'Bitter',serif;
 +
  border-bottom:none;
 +
}
 +
h2,h3,.toc,.show,.hide,.content{
 +
  color:#E6B800;
 +
  font-family:'Bitter',serif;
 +
}
 +
h4,h5,h6, .page{
 +
  color:#222222;
 +
  font-family:'Muli',sans-serif;
 +
}
 +
.toc{
 +
  text-decoration:none;
 +
  display:none;
 +
  width:100%;
 +
  margin:auto;
 +
}
 +
.toc .month-group { margin: 20px 0; }
 +
.toc .month-group p { padding: 0; margin: 0; }
 +
#ftoc{
 +
  color:#666666;
 +
}
 +
ul{
 +
  list-style-type:none;
 +
}
 +
.month-group, a{
 +
  text-decoration:none;
 +
}
 +
.month-group{
 +
  margin-left:20px;
 +
}
 +
.month-group:hover{
 +
  color:#E6B800;
 +
}
 +
.toc a, .toc2 a, #navi a{
 +
  text-decoration:none;
 +
  color:#222222;
 +
  font-weight: bold;
 +
}
 +
.toc a:hover, #ftoc>a:hover, #navi a:hover, .show:hover, .hide:hover, .container>a:hover{
 +
  color:#E6B800 !important;
 +
  cursor:pointer;
 +
}
 +
.toc a, #ftoc, .container a, #navi{
 +
  border-bottom:2px dotted;
 +
}
 +
#navi{
 +
  text-decoration:none;
 +
  font-family:'Bitter',serif;
 +
}
 +
#logo{
 +
  width:120px;
 +
  margin:auto;
 +
  padding:40px;
 +
  border-radius:100%;
 +
}
 +
#banner{
 +
  width:auto;
 +
  height:250px;
 +
  vertical-align:top;
 +
  background-color:#E6B800;
 +
  overflow: hidden
 +
}
 +
.fixed {
 +
  position:fixed !important;
 +
  top:0;
 +
}
 +
.disp{
 +
  display:none;
 +
}
 +
.col {
 +
     width: 20%;
 +
    float: left;
 +
    clear: right;
 +
    margin: 20px 1.66666666666666666666666666%;
 +
    font-family:'Muli',sans-serif;
 +
    font-weight:300;
 +
    font-size:14px;
 +
    color:#222222;
 +
    text-align:left;
 +
}
 +
.clear{
 +
  width:100%;
 +
  clear:both;
 +
}
 +
.container{
 +
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          <h1>High-Value Product</h1>
 
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For our project, we knew we wanted to create a novel product in <u>E. coli</u> that would make our biodegradation pathway economical and desirable - in other words high-value. Terpenoids are one of the largest, most variable classes of molecules. Their uses are numerous, including antibiotics, hormones, carotenoids and anti-malarial compounds. Terpenoid extraction via plant tissues presents challenges, including resulting in low yield and being generally difficult (Martin, 1). Our product is a plant steroid which promotes seed germination, cell division, and increases lateral bud growth (Sigma, 1). Using genetically engineered <u>E. coli</u> is a less-expensive, simpler path to its production. We believe that this product is a high-value alternative to biofuel, cosmetic, and pet food options often created from recycled frying oil. We hope that when created in conjunction with these current recycled products, the amount of frying oil waste disposed of in landfills and sewage can be greatly decreased. <br><br>
 +
 
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In order to increase terpenoid production, <u>E. coli</u> can be fitted with the genes for the mevalonate pathway which directs terpenoid backbone production. Our first plasmid consists of four genes, the pMBI operon, inserted into a puc19 backbone (<a href='#Fig1'>Figure 1</a>). Our second plasmid consists of three genes, the pMevT operon, inserted into a puc19 backbone (<a href='#Fig2'>Figure 2</a>). Together, these two plasmids direct <u>E. coli</u> to metabolize the fatty acids of the frying oil via the mevalonate pathway to create dimethylallyl pyrophosphate (DMAPP). These two plasmids combined were worked on for a majority of the summer.<br><br>
 +
 
 +
 
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Because each of the operons, pMBI and pMevT, require a number of genes to be combined at once, our team chose to use Gibson Assembly as our main method of construction (see more about our Gibson Assembly Protocol <a href='/Team:CSU_Fort_Collins/Protocols=Gibson'>here</a>). This is a challenging method, but is often more successful for inserting multiple pieces into a plasmid backbone at one.
 +
 
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<center><img src='https://static.igem.org/mediawiki/2014/f/ff/Team-CSU_puc19.png' id='Fig1'/><br>Figure 1: Diagram of pMBI in puc19 Plasmid<br><br>
 +
<img src='https://static.igem.org/mediawiki/2014/a/ac/Team-CSU_pMevT.png' id='Fig2'/><br>Figure 2: Diagram of pMevT in puc19 Plasmid<br>
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Revision as of 22:04, 12 October 2014

High-Value Product

Manufacturing a High-Value Product

For our project, we knew we wanted to create a novel product in E. coli that would make our biodegradation pathway economical and desirable - in other words high-value. Terpenoids are one of the largest, most variable classes of molecules. Their uses are numerous, including antibiotics, hormones, carotenoids and anti-malarial compounds. Terpenoid extraction via plant tissues presents challenges, including resulting in low yield and being generally difficult (Martin, 1). Our product is a plant steroid which promotes seed germination, cell division, and increases lateral bud growth (Sigma, 1). Using genetically engineered E. coli is a less-expensive, simpler path to its production. We believe that this product is a high-value alternative to biofuel, cosmetic, and pet food options often created from recycled frying oil. We hope that when created in conjunction with these current recycled products, the amount of frying oil waste disposed of in landfills and sewage can be greatly decreased.

In order to increase terpenoid production, E. coli can be fitted with the genes for the mevalonate pathway which directs terpenoid backbone production. Our first plasmid consists of four genes, the pMBI operon, inserted into a puc19 backbone (Figure 1). Our second plasmid consists of three genes, the pMevT operon, inserted into a puc19 backbone (Figure 2). Together, these two plasmids direct E. coli to metabolize the fatty acids of the frying oil via the mevalonate pathway to create dimethylallyl pyrophosphate (DMAPP). These two plasmids combined were worked on for a majority of the summer.

Because each of the operons, pMBI and pMevT, require a number of genes to be combined at once, our team chose to use Gibson Assembly as our main method of construction (see more about our Gibson Assembly Protocol here). This is a challenging method, but is often more successful for inserting multiple pieces into a plasmid backbone at one.

Figure 1: Diagram of pMBI in puc19 Plasmid


Figure 2: Diagram of pMevT in puc19 Plasmid

Retrieved from "http://2014.igem.org/Team:CSU_Fort_Collins/HVP/"