Team:Tsinghua-A/Wetlab

From 2013.igem.org

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<meta charset="utf-8">
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<title>Tsinghua-A Wetlab</title>
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<meta name="viewport" content="width=device-width, initial-scale=1.0">
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<meta name="description" content="">
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<meta name="author" content="">
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<link rel="stylesheet" type="text/css" href="https://2013.igem.org/Team:Tsinghua-A/css/bs.css?action=raw&ctype=text/css"/>
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Maxim one page bootstrap template
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@2013 by iWebStudio
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</style>
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</head>
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/*
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<body>
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===========================
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<!-- head -->
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CONTENTS:
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<section class="section green1">
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<div class="container">
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<div class="row">
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01 General
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<ul class="ch-grid">
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02 Typography
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    <li>
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03 Non-semantic
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04 Navbar
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05 Page sections
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                <h3><a href="#about">Overview</a></h3>
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06 Footer
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07 Icons
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08 Buttons
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09 Media queries
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===========================
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*/
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                <h3><a href="#life">Construction</a></h3>
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/* flexslider */
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                <h3><a href="#projects">Supplementary text</a></h3>
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                <h3><a href="#publications">Experimental Characterization</a></h3>
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                <h3><a href="#contact">Discussion</a></h3>
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/* =========================================================
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</div>
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01. General
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<h4>Overview</h4>
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<div class="span6 offset1">
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<div>
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<p style="text-align:justify">
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Based on modeling work, we find that negative feedback can contribute to the network’s adaptation to DNA copy number variation. So we analysed the following three-node networks, A and B. The difference between A and B is the output nodes of network A has negative feedback.
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                    </br>
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We transfected this two circuits into mammalian cells (Hela cell). By testing the mean value of EYFP (Enhance Yellow Fluorescent Protein, the output of our circuits), and the relationship between the EYFP and the DNA copy number, we can prove the hypothesis.
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<img src="https://static.igem.org/mediawiki/2013/6/65/Extu1.png" alt="" />
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<img src="https://static.igem.org/mediawiki/2013/3/31/Extu2.png" alt="" />
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<h4>Construction </h4>
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We constructed the following circuit A ,B and C .The circuit A corresponds to the network A ,while the circuit B is the implementation of network B. Circuit C is used as a control design to testify the function of A and B.
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In circuit A, as we can see, the input is miR-21, which can repress the plasmid pz371 and K1116002(The plasmid’s information can be found in parts). K1116002 induced by rtTA and Dox, serves as an auxiliary node, producing the LacI gene to inhibit the expression of EYFP. EYFP(Enhance Yellow Fluorescent Protein )is used as output. Besides, the miR-FF3 restrains the expression of LacI. The reason that we get the most of post-transcriptional control can be seen in Supplementary text.
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                In circuit B, however, the plasmid K1116003 does not have FF3 target, leading to the contrast between circuit A and B. We can see miR-21 can’t target at pZ349 and pZ331 in circuit C , that is, there is no input in circuit C. The miR-21, used to distinguish cancer cell from normal cells ,is endogenous in Hela cell.
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</br>
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<b>Supplementary text</b>
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miRNAs function as posttranscriptional regulators which have distinguished features compared to transcriptional regulators, intervening late in gene expression process, with the capability to counteract variation from the upstream processes (Margaret et al., 2012). Research shows that while conducting experiment on an incoherent feedforward motif in mammalian cells, posttranscriptional regulation results in superior adaptation behavior, higher absolute expression levels and lower intrinsic fluctuations (Bleris et al., 2011). miRNAs can serve as buffers against variation during gene expression; transient increases in transcription factor activity would propagate to increases in target miRNA transcription while would be counteracted by increased miRNA and vice versa. Therefore, under the miRNA posttranscriptional regulation, protein output can be uncoupled from fluctuations in transcription factor concentration or activity (Margaret et al., 2012). miRNAs also possess good stability which, consistent with theoretical constraints, meets the need for enough molecules of a regulator to achieve a small reduction in the noise of a target gene (Lestas et al., 2010).  
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input, button, select, textarea {
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02. Typography
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03. Misc
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/* =========================================================
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04. Navigation
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============================================================ */
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#menu-main {
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/* =========================================================
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05. Page sections
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============================================================ */
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/* === section header === */
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#header-wrapper {
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width: 100%;
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min-height: 600px;
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margin-top: -60px;
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padding-top:160px;
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text-align:center;
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}
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color: #FFFFFF;
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font-size: 4em;
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font-weight: 400;
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margin: 1em 0;
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color: #fff;
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font-family: 'Open Sans', sans serif;
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font-size: 2em;
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font-weight: normal;
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margin: 0.5em 0;
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text-shadow: 1px 1px 1px #333333;
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}
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header .logo {
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text-align:center;
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margin-bottom:30px;
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}
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/* --- Header slider --- */
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#header-wrapper.header-slider {
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background: #444 url(../img/bg/bg-1.jpg) no-repeat center center fixed;
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background-size: cover;
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}
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#main-flexslider ul.slides li p {
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font-size: 72px;
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text-transform: uppercase;
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position: relative;
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margin-bottom: 0px;
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text-align: center;
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line-height: 1;
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color:#fff;
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}
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#main-flexslider.flexslider {
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margin:0;
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/* --- Header subscribe form --- */
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background: rgba(227, 231, 228, 1);
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font-family: 'Open Sans', sans serif;
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font-size: 1.3125em;
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border: 0;
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text-align: center;
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vertical-align: middle;
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margin-bottom:0 !important;
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}
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#header-wrapper input[type=text]:focus {
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background: #43413e;
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color: #eff1ef;
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transition: background 0.25 ease-in;
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-moz-transition: background 0.25 ease-in;
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-webkit-transition: background 0.25 ease-in;
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}
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#header-wrapper input[type=text] {
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height: 2.9em;
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width:50%;
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color: #fff;
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width: 185px;
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height: 2.9em;
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font-size: 1.3125em; /* 21px */
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padding:0.5em;
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letter-spacing: 0.05em;
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margin: 0;
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border: 0;
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text-transform: none;
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border-radius: 3px;
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-moz-border-radius: 3px;
+
-
-webkit-border-radius: 3px;
+
-
}
+
-
#header-wrapper input[type=submit]:hover, #header-wrapper input[type=submit]:active {
+
-
background: #43413e !important;
+
-
}
+
-
 
+
-
 
+
-
section.section {
+
-
padding: 100px 0 0px;
+
-
}
+
-
section.section h4 {
+
-
text-align: center;
+
-
margin-bottom: 40px;
+
-
font-size: 46px;
+
-
font-weight: 700;
+
-
letter-spacing: -1px;
+
-
}
+
-
section.inner {
+
-
padding: 50px 0 50px;
+
-
}
+
-
 
+
-
 
+
-
 
+
-
.section.orange h4 ,.section.green1 h4,.section.green2 h4,.section.green3 h4,.section.green4 h4,.section.green5 h4,.section.green6 h4{
+
-
color: #fcfcfc;
+
-
}
+
-
.section.orange p,.section.green1 p,.section.green2 p,.section.green3 p,.section.green4 p,.section.green5 p,.section.green6 p {
+
-
color: #f7f7f7;
+
-
}
+
-
 
+
-
 
+
-
/* === section spacer === */
+
-
 
+
-
section.spacer {
+
-
padding:90px 0 70px;
+
-
color:#fcfcfc;
+
-
}
+
-
 
+
-
.spacer.green1,.section.green1 {
+
-
background:#1bac91;
+
-
}
+
-
.spacer.green1,.section.green1 h4 {
+
-
color:#fff;
+
-
}
+
-
 
+
-
.spacer.green2,.section.green2 {
+
-
background:#38BE38;
+
-
}
+
-
.spacer.green2,.section.green2 h4 {
+
-
color:#fff;
+
-
}
+
-
.spacer.green3,.section.green3 {
+
-
background:#298B29;
+
-
}
+
-
.spacer.green3,.section.green3 h4 {
+
-
color:#fff;
+
-
}
+
-
 
+
-
.spacer.green4,.section.green4 {
+
-
background:#007B00;
+
-
}
+
-
.spacer.green4,.section.green4 h4 {
+
-
color:#fff;
+
-
}
+
-
.spacer.green5,.section.green5 {
+
-
background:#007200;
+
-
}
+
-
.spacer.green5,.section.green5 h4 {
+
-
color:#fff;
+
-
}
+
-
 
+
-
.spacer.green6,.section.green6 {
+
-
background:#005900;
+
-
}
+
-
.spacer.green6,.section.green6 h4 {
+
-
color:#fff;
+
-
}
+
-
 
+
-
.spacer.yellow,.section.yellow  {
+
-
background:#ffe873;
+
-
}
+
-
.spacer.orange,.section.orange {
+
-
background:#f36f36;
+
-
}
+
-
.spacer.wood,.section.wood  {
+
-
background:#edb887;
+
-
}
+
-
.spacer.blue,.section.blue  {
+
-
background:#CAE1FF;
+
-
}
+
-
 
+
-
section.spacer.bg2 {
+
-
background: url(../img/bg/bg-2.jpg) 50% 0 no-repeat fixed;
+
-
width:100%;
+
-
height:auto;
+
-
margin:0;
+
-
}
+
-
 
+
-
section.spacer.bg3 {
+
-
background: url(../img/bg/bg-3.jpg) 50% 0 no-repeat fixed;
+
-
width:100%;
+
-
height:auto;
+
-
margin:0;
+
-
}
+
-
 
+
-
section.spacer blockquote.large:before {
+
-
color:#eee;
+
-
opacity:0.6;
+
-
}
+
-
 
+
-
section.spacer blockquote cite {
+
-
color:#555;
+
-
}
+
-
 
+
-
 
+
-
 
+
-
/* === section works === */
+
-
 
+
-
#works h4 {
+
-
margin-bottom:20px;
+
-
}
+
-
 
+
-
#portfolio {
+
-
padding-bottom: 0;
+
-
}
+
-
/* === portfolio filter === */
+
-
 
+
-
#filters {
+
-
margin-bottom: 0px;
+
-
text-align: center;
+
-
display: block;
+
-
float: none;
+
-
z-index: 2;
+
-
position: relative;
+
-
}
+
-
#filters ul {
+
-
margin: 0 0 20px;
+
-
list-style: none;
+
-
padding: 0;
+
-
}
+
-
#filters ul li {
+
-
display: inline-block;
+
-
}
+
-
#filters ul li a {
+
-
display: block;
+
-
float: left;
+
-
padding: 2px 5px;
+
-
color: inherit;
+
-
margin-right: 5px;
+
-
margin-bottom: 5px;
+
-
font-weight: 600;
+
-
}
+
-
#filters ul li a:hover,#filters ul li a:focus {
+
-
text-decoration:none;
+
-
outline:0;
+
-
}
+
-
#filters ul li a h5 {
+
-
font-size: 14px;
+
-
padding: 5px 10px 5px;
+
-
border: 2px solid #f2f2f2;
+
-
}
+
-
 
+
-
#filters ul li a:hover h5, #filters ul li a.active h5 {
+
-
color:#fcfcfc;
+
-
}
+
-
 
+
-
/* === portfolio filter2 === */
+
-
 
+
-
#filters2 {
+
-
margin-bottom: 0px;
+
-
text-align: center;
+
-
display: block;
+
-
float: none;
+
-
z-index: 2;
+
-
position: relative;
+
-
}
+
-
#filters2 ul {
+
-
margin: 0 0 20px;
+
-
list-style: none;
+
-
padding: 0;
+
-
}
+
-
#filters2 ul li {
+
-
display: inline-block;
+
-
}
+
-
#filters2 ul li a {
+
-
display: block;
+
-
float: left;
+
-
padding: 2px 5px;
+
-
color: inherit;
+
-
margin-right: 5px;
+
-
margin-bottom: 5px;
+
-
font-weight: 600;
+
-
}
+
-
#filters2 ul li a:hover,#filters2 ul li a:focus {
+
-
text-decoration:none;
+
-
outline:0;
+
-
}
+
-
#filters2 ul li a h5 {
+
-
font-size: 14px;
+
-
padding: 5px 10px 5px;
+
-
border: 2px solid #f2f2f2;
+
-
}
+
-
 
+
-
#filters2 ul li a:hover h5, #filters2 ul li a.active h5 {
+
-
color:#fcfcfc;
+
-
background:#ee6363;
+
-
}
+
-
 
+
-
 
+
-
 
+
-
 
+
-
/* === portfolio wrap - grid === */
+
-
#portfolio-wrap {
+
-
position: relative;
+
-
padding: 0;
+
-
width: 100.1%;
+
-
margin: 0 auto;
+
-
display: block;
+
-
}
+
-
#portfolio-wrap .grid {
+
-
margin: 0;
+
-
overflow: hidden;
+
-
line-height: 0;
+
-
width: 20%;
+
-
border:0;
+
-
padding: 0;
+
-
}
+
-
.portfolio-item.current {
+
-
z-index: 101;
+
-
}
+
-
.portfolio-item {
+
-
padding: 0;
+
-
position: relative;
+
-
overflow: hidden;
+
-
}
+
-
.portfolio-item .portfolio-image {
+
-
overflow: hidden;
+
-
display: block;
+
-
position: relative;
+
-
}
+
-
.portfolio-item .portfolio-image img {
+
-
width: 100%;
+
-
height: auto;
+
-
}
+
-
.portfolio .portfolio-overlay {
+
-
width: 100%;
+
-
height: 100%;
+
-
top: 0;
+
-
left: 0;
+
-
position: absolute;
+
-
display: block;
+
-
z-index: 4;
+
-
opacity: 0;
+
-
-moz-opacity: 0;
+
-
filter: alpha(opacity=0);
+
-
-webkit-transition: all 0.3s ease-in-out;
+
-
-moz-transition: all 0.3s ease-in-out;
+
-
-o-transition: all 0.3s ease-in-out;
+
-
-ms-transition: all 0.3s ease-in-out;
+
-
transition: all 0.3s ease-in-out;
+
-
}
+
-
.portfolio > a, .portfolio > a:hover {
+
-
color: #ffffff;
+
-
}
+
-
.portfolio-item .portfolio > a:hover > .portfolio-overlay {
+
-
opacity: 0.5;
+
-
-moz-opacity: 0.5;
+
-
filter: alpha(opacity=50);
+
-
}
+
-
.portfolio > a img {
+
-
-webkit-transition: all 0.3s ease-in-out;
+
-
-moz-transition: all 0.3s ease-in-out;
+
-
-o-transition: all 0.3s ease-in-out;
+
-
-ms-transition: all 0.3s ease-in-out;
+
-
transition: all 0.3s ease-in-out;
+
-
}
+
-
.portfolio > a:hover img {
+
-
-webkit-transition: all 0.3s ease-in-out;
+
-
-moz-transition: all 0.3s ease-in-out;
+
-
-o-transition: all 0.3s ease-in-out;
+
-
-ms-transition: all 0.3s ease-in-out;
+
-
transition: all 0.3s ease-in-out;
+
-
}
+
-
.portfolio .thumb-info {
+
-
position: absolute;
+
-
width: 100%;
+
-
height: 100%;
+
-
opacity: 0;
+
-
-moz-opacity: 0;
+
-
filter: alpha(opacity=0);
+
-
-webkit-transition: all 0.3s ease-in-out;
+
-
-moz-transition: all 0.3s ease-in-out;
+
-
-o-transition: all 0.3s ease-in-out;
+
-
-ms-transition: all 0.3s ease-in-out;
+
-
transition: all 0.3s ease-in-out;
+
-
}
+
-
.portfolio a:hover .portfolio-overlay .thumb-info {
+
-
opacity: 1;
+
-
-moz-opacity: 1;
+
-
filter: alpha(opacity=100);
+
-
}
+
-
.portfolio-overlay .thumb-info i {
+
-
top: 50%;
+
-
}
+
-
.portfolio-overlay .thumb-info h5 {
+
-
top: 20%;
+
-
font-size: 18px;
+
-
}
+
-
.portfolio-overlay .thumb-info p {
+
-
top: 50%;
+
-
font-size: 13px;
+
-
}
+
-
.portfolio-overlay .thumb-info h5 {
+
-
color: #ffffff;
+
-
width: 100%;
+
-
position: absolute;
+
-
text-align: center;
+
-
}
+
-
.portfolio-overlay .thumb-info p {
+
-
color: #ffffff;
+
-
width: 100%;
+
-
position: absolute;
+
-
text-align: center;
+
-
font-weight: bold;
+
-
}
+
-
.portfolio-overlay .thumb-info i {
+
-
color: #ffffff;
+
-
width: 100%;
+
-
position: absolute;
+
-
text-align: center;
+
-
display: block;
+
-
}
+
-
/* === Isotope CSS3 transitions === */
+
-
 
+
-
.isotope, .isotope .isotope-item {
+
-
-webkit-transition-duration: 0.8s;
+
-
-moz-transition-duration: 0.8s;
+
-
-ms-transition-duration: 0.8s;
+
-
-o-transition-duration: 0.8s;
+
-
transition-duration: 0.8s;
+
-
}
+
-
.isotope {
+
-
-webkit-transition-property: height, width;
+
-
-moz-transition-property: height, width;
+
-
-ms-transition-property: height, width;
+
-
-o-transition-property: height, width;
+
-
transition-property: height, width;
+
-
}
+
-
.isotope .isotope-item {
+
-
-webkit-transition-property: -webkit-transform, opacity;
+
-
-moz-transition-property: -moz-transform, opacity;
+
-
-ms-transition-property: -ms-transform, opacity;
+
-
-o-transition-property: -o-transform, opacity;
+
-
transition-property: transform, opacity;
+
-
}
+
-
/**** disabling Isotope CSS3 transitions ****/
+
-
 
+
-
.isotope.no-transition, .isotope.no-transition .isotope-item, .isotope .isotope-item.no-transition {
+
-
-webkit-transition-duration: 0s;
+
-
-moz-transition-duration: 0s;
+
-
-ms-transition-duration: 0s;
+
-
-o-transition-duration: 0s;
+
-
transition-duration: 0s;
+
-
}
+
-
 
+
-
 
+
-
/* === section services === */
+
-
 
+
-
section#services h2 {
+
-
font-size: 22px;
+
-
margin-bottom: 15px;
+
-
margin-top: 10px;
+
-
text-align:center;
+
-
}
+
-
section#services p {
+
-
margin:10px;
+
-
}
+
-
 
+
-
 
+
-
section#services .service-box {
+
-
text-align: center;
+
-
padding: 20px;
+
-
-webkit-transition: all 0.3s ease-in-out;
+
-
-moz-transition: all 0.3s ease-in-out;
+
-
-o-transition: all 0.3s ease-in-out;
+
-
-ms-transition: all 0.3s ease-in-out;
+
-
transition: all 0.3s ease-in-out;
+
-
}
+
-
section#services .span3:hover {
+
-
background:#e3e3e3;
+
-
}
+
-
 
+
-
section#services .service-box h2 {
+
-
color: #f7f7f7;
+
-
font-weight: 700;
+
-
}
+
-
 
+
-
section#services.section.orange .service-box:hover {
+
-
background:#f36518;
+
-
}
+
-
 
+
-
 
+
-
/* === section blog === */
+
-
 
+
-
section#blog {
+
-
background: #f2f2f2;
+
-
}
+
-
 
+
-
.home-post {
+
-
background:#fff;
+
-
}
+
</div>
 +
</section>
-
.home-post:hover {
+
<!-- end section: projects -->
-
box-shadow: 0 0 16px rgba(0, 0, 0, 0.3);
+
<section id="character" class="section green4">
-
-moz-box-shadow: 0 0 16px rgba(0, 0, 0, 0.3);
+
<div class="container">
-
-webkit-box-shadow: 0 0 16px rgba(0, 0, 0, 0.3);
+
<h4>Experimental Characterization</h4>
-
-webkit-transition: all 0.4s ease-in-out;
+
<div class="row">
-
-moz-transition: all 0.4s ease-in-out;
+
-
-o-transition: all 0.4s ease-in-out;
+
<div>
-
-ms-transition: all 0.4s ease-in-out;
+
-
transition: all 0.4s ease-in-out;
+
<p >
-
}
+
  We took advantage of another fluorescent protein(mkate ) as reference gene, which has no influence in our design. Published literature generally supports the view that in transient transfections, fluorescence depends linearly on the copy number of transfected plasmids (Tseng et al, 1997; Pollard et al, 1998;Cohenet al,2009; Schwakeet al, 2010). While strictly speaking, this reporter level also depends on many other potentially fluctuating parameters such as global synthesis and degradation rates(Leonidas Bleris et al,2011),it is more legitimately use the normalized quotient to instead of the value of EYFP.
-
 
+
                  </br></br>
-
 
+
  <img src="https://static.igem.org/mediawiki/2013/8/87/Extu6.jpg" alt="" style="margin:auto;display:block;text-align:center"/>
-
.home-post .entry-content {
+
  </br>
-
padding:20px;
+
  Apparently, the output of circuit C is lower than circuit B .Having the negative feedback compared with circuit B, the expression of EYFP in circuit A is strongest.  
-
}
+
  </br>
-
.home-post .entry-content h5 a {
+
  </br>
-
font-size:22px;
+
                  Then we analysed the output of constructed designs varies with the DNA copy number. Facing with the difficulty of counting the copy number directly, we employed the reference gene to reflect .We think the copy number is high when the expression intensity of mkate is strong. One hundred thousand positive Hela cells was collected to obtain the relationship between EYFP and make.
-
}
+
  </br></br>
-
.home-post .entry-content h5 a:hover {
+
      <img src="https://static.igem.org/mediawiki/2013/e/e1/Extu7.jpg" alt="" style="margin:auto;display:block;text-align:center"/>
-
color:#312f2b;
+
  </br>
-
text-decoration:none;
+
  From the figure 2, we learned with the increase of the expression intensity of makte, the specific valve of EYFP and mkate decreases. In another word, the circuit A’s output reaches saturation fast with the increase of copy number. We came to a conclusion circuit A’s adaptation to DNA copy number is higher than circuit B’s. So, the negative feedback works.
-
}
+
-
.home-post .entry-content a.more {
+
</p>
-
font-size:13px;
+
</div>
-
}
+
</div>
-
.home-post .post-meta {
+
-
padding:20px;
+
</div>
-
overflow:hidden;
+
-
color:#fcfcfc;
+
-
}
+
-
.home-post .post-meta i {
+
-
float:left;
+
-
margin-right:20px;
+
-
}
+
-
.home-post .post-meta span {
+
-
margin-top:10px;
+
-
}
+
-
.home-post .post-meta span.date {
+
-
font-weight:700;
+
-
font-size:13px;
+
-
float:left;
+
-
}
+
-
.home-post .post-meta span.tags {
+
-
font-weight:700;
+
-
font-size:13px;
+
-
float:right;
+
-
color:#ddd;
+
-
}
+
-
.home-post .post-meta span.tags a {
+
-
color:#fcfcfc;
+
-
}
+
-
 
+
-
 
+
-
 
+
-
/* === section about === */
+
-
 
+
-
section#about {
+
-
background:#f2f2f2;
+
-
}
+
-
 
+
-
.people {
+
-
text-align:center;
+
-
}
+
-
 
+
-
.people h3 {
+
-
margin-bottom:10px;
+
-
font-weight:500;
+
-
letter-spacing:-1px;
+
-
}
+
-
 
+
-
.team-thumb {
+
-
margin-top: 26px;
+
-
text-align:center;
+
-
}
+
-
 
+
-
/* (spacer) latest tweet */
+
-
#tweet{
+
-
text-align:center;
+
-
margin:20px 0 0;
+
-
font-size:20px;
+
-
color:#fff;
+
-
}
+
-
 
+
-
#tweet ul{
+
-
float:left;
+
-
width:100%;
+
-
text-align:center;
+
-
list-style:none;
+
-
margin:0;
+
-
padding:0;
+
-
}
+
-
 
+
-
#tweet ul li{
+
-
display: inline-block;
+
-
list-style:none;
+
-
margin:0 0 20px;
+
-
padding:0 0 0 34px;
+
-
font-size:20px;
+
-
line-height:1.5em;
+
-
}
+
-
 
+
-
#tweet ul li:before {
+
-
font-family: 'FontAwesome';
+
-
speak: none;
+
-
font-style: normal;
+
-
font-weight: normal;
+
-
font-variant: normal;
+
-
text-transform: none;
+
-
line-height: 1;
+
-
-webkit-font-smoothing: antialiased;
+
-
content: "\f099";
+
-
margin-right:20px;
+
-
}
+
-
 
+
-
/* === Contact section === */
+
-
 
+
-
 
+
-
/* Contact Form */
+
-
 
+
-
.validation {
+
-
display:none;
+
-
margin: -10px 0 20px;
+
-
color:#fff;
+
-
font-weight:700;
+
-
font-size:13px;
+
-
}
+
-
 
+
-
#sendmessage {
+
-
border:1px solid #fff;
+
-
display:none;
+
-
text-align:center;
+
-
padding:15px 12px 15px;
+
-
margin:10px 0;
+
-
font-weight:600;
+
-
margin-bottom:30px;
+
-
}
+
-
 
+
-
#sendmessage.show,.show  {
+
-
display:block;
+
-
}
+
-
 
+
-
#contact-form {
+
-
position: relative;
+
-
z-index: 999;
+
-
}
+
-
 
+
-
#contact-form input[type=text], .contactForm textarea {
+
-
background: rgba(227, 231, 228, 1);
+
-
font-family: 'Open Sans', sans serif;
+
-
border: 0;
+
-
font-size:16px;
+
-
text-align: left;
+
-
vertical-align: middle;
+
-
}
+
-
#contact-form input[type=text]:focus, .contactForm textarea:focus {
+
-
background: rgba(101, 106, 100, 1);
+
-
color: #eff1ef;
+
-
box-shadow: none;
+
-
-moz-box-shadow: none;
+
-
-webkit-box-shadow: none;
+
-
transition: background 0.25 ease-in;
+
-
-moz-transition: background 0.25 ease-in;
+
-
-webkit-transition: background 0.25 ease-in;
+
-
}
+
-
#contact-form input[type=text] {
+
-
height: 60px;
+
-
margin-bottom:30px;
+
-
}
+
-
#contact-form input[type=submit] {
+
-
color: #fff;
+
-
width: 185px;
+
-
height: 60px;
+
-
text-shadow: none;
+
-
font-size: 16px;
+
-
padding:0.5em;
+
-
letter-spacing: 0.05em;
+
-
margin: 0 0 20px 0;
+
-
display: block;
+
-
border: 0;
+
-
text-transform: none;
+
-
border-radius: 3px;
+
-
-moz-border-radius: 3px;
+
-
-webkit-border-radius: 3px;
+
-
box-shadow: none;
+
-
-moz-box-shadow: none;
+
-
-webkit-box-shadow: none;
+
-
}
+
-
#contact-form input[type=submit]:hover, #contact-form input[type=submit]:active {
+
-
background: #43413e !important;
+
-
}
+
-
#contact-form textarea {
+
-
padding-top: 1em;
+
-
}
+
-
div.cform-response-output {
+
-
max-width: 60%;
+
-
text-align: center;
+
-
margin-left: 40% !important;
+
-
margin-top: 0.5em !important;
+
-
padding: 0.5em !important;
+
-
border-radius: 4px;
+
-
-moz-border-radius: 4px;
+
-
-webkit-border-radius: 4px;
+
-
}
+
-
.cform-not-valid-tip {
+
-
color: #888;
+
-
border: 1px dotted #ad3729 !important;
+
-
width: 100% !important;
+
-
left: 0 !important;
+
-
padding: 0.5em !important;
+
-
font-family: 'Open Sans', sans-serif;
+
-
box-sizing: border-box;
+
-
-moz-box-sizing: border-box;
+
-
-webkit-box-sizing: border-box;
+
-
border-radius: 4px;
+
-
-moz-border-radius: 4px;
+
-
-webkit-border-radius: 4px;
+
-
}
+
-
 
+
-
 
+
-
 
+
-
/* =========================================================
+
-
06. Footer
+
-
============================================================ */
+
-
 
+
-
footer {
+
-
background: #2b2b2b;
+
-
text-align: center;
+
-
color: #5e5e5e;
+
-
padding:50px 0 50px;
+
-
}
+
-
 
+
-
footer ul.social-networks {
+
-
list-style:none;
+
-
}
+
-
 
+
-
footer ul.social-networks li {
+
-
display:inline;
+
-
}
+
-
 
+
-
footer ul.social-networks li i {
+
-
display:inline-block;
+
-
}
+
-
 
+
-
footer ul.social-networks li a {
+
-
color: #5e5e5e;
+
-
margin: 0 5px;
+
-
}
+
-
 
+
-
footer ul.social-networks li a:hover {
+
-
text-decoration:none;
+
-
-webkit-transition: all 0.4s ease-in-out;
+
-
-moz-transition: all 0.4s ease-in-out;
+
-
-o-transition: all 0.4s ease-in-out;
+
-
-ms-transition: all 0.4s ease-in-out;
+
-
transition: all 0.4s ease-in-out;
+
-
}
+
-
 
+
-
footer ul.social-networks li i.icon-bgdark {
+
-
background:#414141;
+
-
color: #656565;
+
-
}
+
-
 
+
-
 
+
-
footer ul.social-networks li a i.icon-group:hover {
+
-
background: #ba7b3d;
+
-
color: #fff;
+
-
}
+
-
 
+
-
footer ul.social-networks li a i.icon-dashboard:hover {
+
-
background: #5ad2eb;
+
-
color: #fff;
+
-
}
+
-
footer ul.social-networks li a i.icon-dribbble:hover {
+
-
background: #f57b71;
+
-
color: #fff;
+
-
}
+
-
footer ul.social-networks li a i.icon-pinterest:hover {
+
-
background: #d84848;
+
-
color: #fff;
+
-
}
+
-
 
+
-
 
+
-
 
+
-
 
+
-
/* =========================================================
+
-
07. Icons
+
-
============================================================ */
+
-
 
+
-
/* bg */
+
-
i.icon-bglight {
+
-
color: #444;
+
-
background: #f7f7f7;
+
-
}
+
-
i.icon-bglight.active {
+
-
color: #fff;
+
-
}
+
-
 
+
-
i.icon-bglight.active:hover{
+
-
color: #444;
+
-
background: #f7f7f7;
+
-
}
+
-
 
+
-
i.icon-bgdark {
+
-
color: #f7f7f7;
+
-
background: #333;
+
-
}
+
-
i.icon-bgdark:hover {
+
-
color: #fff;
+
-
}
+
-
i.icon-bgdark.active:hover {
+
-
color: #f7f7f7;
+
-
background: #333;
+
-
}
+
-
 
+
-
/* square */
+
-
i.icon-square {
+
-
display: inline-block;
+
-
text-align: center;
+
-
width: 24px !important;
+
-
height: 24px !important;
+
-
line-height: 24px !important;
+
-
}
+
-
i.icon-square.icon-2x{
+
-
width: 48px !important;
+
-
height: 48px !important;
+
-
line-height: 48px !important;
+
-
}
+
-
i.icon-square.icon-3x{
+
-
width: 72px !important;
+
-
height: 72px !important;
+
-
line-height: 72px !important;
+
-
}
+
-
i.icon-square.icon-4x{
+
-
width: 96px !important;
+
-
height: 96px !important;
+
-
line-height: 96px !important;
+
-
}
+
-
i.icon-square.icon-5x{
+
-
width: 120px !important;
+
-
height: 120px !important;
+
-
line-height: 120px !important;
+
-
}
+
-
 
+
-
/* rounded */
+
-
i.icon-rounded {
+
-
display: inline-block;
+
-
border-radius: 6px !important;
+
-
-moz-border-radius: 6px !important;
+
-
-webkit-border-radius: 6px !important;
+
-
-o-border-radius: 6px !important;
+
-
-ms-border-radius: 6px !important;
+
-
text-align: center;
+
-
width: 24px !important;
+
-
height: 24px !important;
+
-
line-height: 24px !important;
+
-
}
+
-
 
+
-
.icon-rounded.icon-2x{
+
-
width: 48px !important;
+
-
height: 48px !important;
+
-
line-height: 48px !important;
+
-
}
+
-
.icon-rounded.icon-3x{
+
-
width: 72px !important;
+
-
height: 72px !important;
+
-
line-height: 72px !important;
+
-
border-radius: 8px !important;
+
-
-moz-border-radius: 8px !important;
+
-
-webkit-border-radius: 8px !important;
+
-
-o-border-radius: 8px !important;
+
-
-ms-border-radius: 8px !important;
+
-
}
+
-
.icon-rounded.icon-4x{
+
-
width: 96px !important;
+
-
height: 96px !important;
+
-
line-height: 96px !important;
+
-
border-radius: 10px !important;
+
-
-moz-border-radius: 10px !important;
+
-
-webkit-border-radius: 10px !important;
+
-
-o-border-radius: 10px !important;
+
-
-ms-border-radius: 10px !important;
+
-
}
+
-
.icon-rounded.icon-5x{
+
-
width: 120px !important;
+
-
height: 120px !important;
+
-
line-height: 120px !important;
+
-
border-radius: 12px !important;
+
-
-moz-border-radius: 12px !important;
+
-
-webkit-border-radius: 12px !important;
+
-
-o-border-radius: 12px !important;
+
-
-ms-border-radius: 12px !important;
+
-
}
+
-
 
+
-
 
+
-
/* circled */
+
-
i.icon-circled {
+
-
display: inline-block;
+
-
border-radius: 1000px !important;
+
-
-moz-border-radius: 1000px !important;
+
-
-webkit-border-radius: 1000px !important;
+
-
-o-border-radius: 1000px !important;
+
-
-ms-border-radius: 1000px !important;
+
-
text-align: center;
+
-
width: 24px !important;
+
-
height: 24px !important;
+
-
line-height: 24px !important;
+
-
}
+
-
 
+
-
.icon-circled.icon-2x{
+
-
width: 48px !important;
+
-
height: 48px !important;
+
-
line-height: 48px !important;
+
-
}
+
-
.icon-circled.icon-3x{
+
-
width: 72px !important;
+
-
height: 72px !important;
+
-
line-height: 72px !important;
+
-
}
+
-
.icon-circled.icon-4x{
+
-
width: 96px !important;
+
-
height: 96px !important;
+
-
line-height: 96px !important;
+
-
}
+
-
.icon-circled.icon-5x{
+
-
width: 120px !important;
+
-
height: 120px !important;
+
-
line-height: 120px !important;
+
-
}
+
-
 
+
-
i.icon-white {
+
-
color:#fff;
+
-
}
+
-
 
+
-
 
+
-
/* more icon sizes for font-awesome icons */
+
-
 
+
-
.icon-6x {
+
-
  font-size: 6em;
+
-
}
+
-
.icon-6x.icon-border {
+
-
  border-width: 6px;
+
-
  -webkit-border-radius: 8px;
+
-
  -moz-border-radius: 8px;
+
-
  border-radius: 8px;
+
-
}
+
-
 
+
-
.icon-7x {
+
-
  font-size: 7em;
+
-
}
+
-
.icon-6x.icon-border {
+
-
  border-width: 6px;
+
-
  -webkit-border-radius: 8px;
+
-
  -moz-border-radius: 8px;
+
-
  border-radius: 8px;
+
-
}
+
-
 
+
-
.icon-8x {
+
-
  font-size: 8em;
+
-
}
+
-
.icon-6x.icon-border {
+
-
  border-width: 6px;
+
-
  -webkit-border-radius: 8px;
+
-
  -moz-border-radius: 8px;
+
-
  border-radius: 8px;
+
-
}
+
-
 
+
-
.icon-9x {
+
-
  font-size: 9em;
+
-
}
+
-
.icon-6x.icon-border {
+
-
  border-width: 6px;
+
-
  -webkit-border-radius: 8px;
+
-
  -moz-border-radius: 8px;
+
-
  border-radius: 8px;
+
-
}
+
-
.icon-10x {
+
-
  font-size: 10em;
+
-
}
+
-
.icon-6x.icon-border {
+
-
  border-width: 6px;
+
-
  -webkit-border-radius: 8px;
+
-
  -moz-border-radius: 8px;
+
-
  border-radius: 8px;
+
-
}
+
-
 
+
-
 
+
-
 
+
-
/* =========================================================
+
-
08. Buttons
+
-
============================================================ */
+
-
+
-
.btn {
+
-
color: #fff;
+
-
text-shadow: none;
+
-
font-size: 16px;
+
-
padding: 11px 19px;
+
-
letter-spacing: 0.05em;
+
-
border: 0;
+
-
text-transform: none;
+
-
border-radius: 3px;
+
-
-moz-border-radius: 3px;
+
-
-webkit-border-radius: 3px;
+
-
box-shadow: none;
+
-
-moz-box-shadow: none;
+
-
-webkit-box-shadow: none;
+
-
  background-color: #545454;
+
-
  border-color: none;
+
-
  *background-color: #545454;
+
-
  background-image: none;
+
-
  /* Darken IE7 buttons by default so they stand out more given they won't have borders */
+
-
  filter: none;
+
-
  border: none;
+
-
  *border: 0;
+
-
  border-bottom-color: none;
+
-
}
+
-
.btn:link, .btn:visited {
+
-
color: #fff;
+
-
}
+
-
.btn:hover {
+
-
background: #333 !important;
+
-
}
+
-
.btn:hover,
+
-
.btn:focus,
+
-
.btn:active,
+
-
.btn.active,
+
-
.btn.disabled,
+
-
.btn[disabled] {
+
-
  color: #fff;
+
-
  background-color: #333;
+
-
  *background-color: #333;
+
-
}
+
-
 
+
-
 
+
-
.btn.btn-theme:hover {
+
-
background: #333 !important;
+
-
}
+
-
 
+
-
/* size */
+
-
.btn-large {
+
-
  padding: 18px 25px;
+
-
  font-size: 24px;
+
-
  -webkit-border-radius: 0;
+
-
  -moz-border-radius: 0;
+
-
  border-radius: 0;
+
-
}
+
-
 
+
-
.btn-large [class^="icon-"],
+
-
.btn-large [class*=" icon-"] {
+
-
  margin-top: 4px;
+
-
}
+
-
 
+
-
.btn-small {
+
-
  padding: 6px 13px;
+
-
  font-size: 11.9px;
+
-
  -webkit-border-radius: 0;
+
-
  -moz-border-radius: 0;
+
-
  border-radius: 0;
+
-
}
+
-
 
+
-
.btn-small [class^="icon-"],
+
-
.btn-small [class*=" icon-"] {
+
-
  margin-top: 0;
+
-
}
+
-
 
+
-
.btn-mini [class^="icon-"],
+
-
.btn-mini [class*=" icon-"] {
+
-
  margin-top: -1px;
+
-
}
+
-
 
+
-
.btn-mini {
+
-
  padding: 0 6px;
+
-
  font-size: 10.5px;
+
-
  -webkit-border-radius: 0;
+
-
  -moz-border-radius: 0;
+
-
  border-radius: 0;
+
-
}
+
-
 
+
-
/* end size */
+
-
 
+
-
.btn-primary {
+
-
  color: #ffffff;
+
-
  text-shadow: none;
+
-
  background-color: #09828c;
+
-
  *background-color: #09828c;
+
-
  background-image: none;
+
-
  border-color: none;
+
-
  filter:none;
+
-
}
+
-
 
+
-
.btn-primary:hover,
+
-
.btn-primary:focus,
+
-
.btn-primary:active,
+
-
.btn-primary.active,
+
-
.btn-primary.disabled,
+
-
.btn-primary[disabled] {
+
-
  color: #ffffff;
+
-
  background: #065158;
+
-
  *background-color: #065158;
+
-
}
+
-
 
+
-
.btn-primary:active,
+
-
.btn-primary.active {
+
-
  background-color: #065158 \9;
+
-
}
+
-
 
+
-
.btn-warning {
+
-
  color: #ffffff;
+
-
  text-shadow: none;
+
-
  background-color: #ffe873;
+
-
  *background-color: #ffe873;
+
-
  background-image: none;
+
-
  border-color: none;
+
-
  filter:none;
+
-
}
+
-
 
+
-
.btn-warning:hover,
+
-
.btn-warning:focus,
+
-
.btn-warning:active,
+
-
.btn-warning.active,
+
-
.btn-warning.disabled,
+
-
.btn-warning[disabled] {
+
-
  color: #ffffff;
+
-
  background: #b88f22;
+
-
  *background: #b88f22;
+
-
}
+
-
 
+
-
.btn-warning:active,
+
-
.btn-warning.active {
+
-
  background: #b88f22 \9;
+
-
}
+
-
 
+
-
 
+
-
.btn-danger {
+
-
  color: #ffffff;
+
-
  text-shadow: none;
+
-
  background-color: #f36f36;
+
-
  *background-color: #f36f36;
+
-
  background-image: none;
+
-
  border-color: none;
+
-
  filter:none;
+
-
}
+
-
 
+
-
.btn-danger:hover,
+
-
.btn-danger:focus,
+
-
.btn-danger:active,
+
-
.btn-danger.active,
+
-
.btn-danger.disabled,
+
-
.btn-danger[disabled] {
+
-
  color: #ffffff;
+
-
  background: #db5f58;
+
-
  *background: #db5f58;
+
-
}
+
-
 
+
-
.btn-danger:active,
+
-
.btn-danger.active {
+
-
  background: #db5f58 \9;
+
-
}
+
-
 
+
-
.btn-success {
+
-
  color: #ffffff;
+
-
  text-shadow: none;
+
-
  background-color: #1bac91;
+
-
  *background-color: #1bac91;
+
-
  background-image: none;
+
-
  border-color: none;
+
-
  filter:none;
+
-
}
+
-
 
+
-
.btn-success:hover,
+
-
.btn-success:focus,
+
-
.btn-success:active,
+
-
.btn-success.active,
+
-
.btn-success.disabled,
+
-
.btn-success[disabled] {
+
-
  color: #ffffff;
+
-
  background: #55ba93;
+
-
  *background: #55ba93;
+
-
}
+
-
 
+
-
.btn-success:active,
+
-
.btn-success.active {
+
-
  background: #55ba93 \9;
+
-
}
+
-
 
+
-
.btn-info {
+
-
  color: #ffffff;
+
-
  text-shadow: none;
+
-
  background-color: #4dbdc6;
+
-
  *background-color: #4dbdc6;
+
-
  background-image: none;
+
-
  border-color: none;
+
-
  filter:none;
+
-
}
+
-
 
+
-
.btn-info:hover,
+
-
.btn-info:focus,
+
-
.btn-info:active,
+
-
.btn-info.active,
+
-
.btn-info.disabled,
+
-
.btn-info[disabled] {
+
-
  color: #ffffff;
+
-
  background: #6a9ad0;
+
-
  *background: #6a9ad0;
+
-
}
+
-
 
+
-
.btn-info:active,
+
-
.btn-info.active {
+
-
  background: #6a9ad0 \9;
+
-
}
+
-
 
+
-
.btn-inverse {
+
-
  color: #ffffff;
+
-
  text-shadow: none;
+
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Due to some restrictions in wetlab, we only finished the above-mentioned experiment. We found that the number of Hela cells who possesses high copy number is  comparatively low. We also noticed the circuit C’s output is higher than expected in Figure 1.This may cause wrong judge when use the design to detect miR-21. Some measures will be taken to solve this question.
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        Besides, we are going to endeavor to construct the other networks mentioned in modeling work.
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Revision as of 04:51, 27 September 2013

Tsinghua-A Wetlab

Overview

Based on modeling work, we find that negative feedback can contribute to the network’s adaptation to DNA copy number variation. So we analysed the following three-node networks, A and B. The difference between A and B is the output nodes of network A has negative feedback.
We transfected this two circuits into mammalian cells (Hela cell). By testing the mean value of EYFP (Enhance Yellow Fluorescent Protein, the output of our circuits), and the relationship between the EYFP and the DNA copy number, we can prove the hypothesis.

Construction

We constructed the following circuit A ,B and C .The circuit A corresponds to the network A ,while the circuit B is the implementation of network B. Circuit C is used as a control design to testify the function of A and B.
In circuit A, as we can see, the input is miR-21, which can repress the plasmid pz371 and K1116002(The plasmid’s information can be found in parts). K1116002 induced by rtTA and Dox, serves as an auxiliary node, producing the LacI gene to inhibit the expression of EYFP. EYFP(Enhance Yellow Fluorescent Protein )is used as output. Besides, the miR-FF3 restrains the expression of LacI. The reason that we get the most of post-transcriptional control can be seen in Supplementary text.
In circuit B, however, the plasmid K1116003 does not have FF3 target, leading to the contrast between circuit A and B. We can see miR-21 can’t target at pZ349 and pZ331 in circuit C , that is, there is no input in circuit C. The miR-21, used to distinguish cancer cell from normal cells ,is endogenous in Hela cell.

Supplementary text

miRNAs function as posttranscriptional regulators which have distinguished features compared to transcriptional regulators, intervening late in gene expression process, with the capability to counteract variation from the upstream processes (Margaret et al., 2012). Research shows that while conducting experiment on an incoherent feedforward motif in mammalian cells, posttranscriptional regulation results in superior adaptation behavior, higher absolute expression levels and lower intrinsic fluctuations (Bleris et al., 2011). miRNAs can serve as buffers against variation during gene expression; transient increases in transcription factor activity would propagate to increases in target miRNA transcription while would be counteracted by increased miRNA and vice versa. Therefore, under the miRNA posttranscriptional regulation, protein output can be uncoupled from fluctuations in transcription factor concentration or activity (Margaret et al., 2012). miRNAs also possess good stability which, consistent with theoretical constraints, meets the need for enough molecules of a regulator to achieve a small reduction in the noise of a target gene (Lestas et al., 2010).

Experimental Characterization

We took advantage of another fluorescent protein(mkate ) as reference gene, which has no influence in our design. Published literature generally supports the view that in transient transfections, fluorescence depends linearly on the copy number of transfected plasmids (Tseng et al, 1997; Pollard et al, 1998;Cohenet al,2009; Schwakeet al, 2010). While strictly speaking, this reporter level also depends on many other potentially fluctuating parameters such as global synthesis and degradation rates(Leonidas Bleris et al,2011),it is more legitimately use the normalized quotient to instead of the value of EYFP.


Apparently, the output of circuit C is lower than circuit B .Having the negative feedback compared with circuit B, the expression of EYFP in circuit A is strongest.

Then we analysed the output of constructed designs varies with the DNA copy number. Facing with the difficulty of counting the copy number directly, we employed the reference gene to reflect .We think the copy number is high when the expression intensity of mkate is strong. One hundred thousand positive Hela cells was collected to obtain the relationship between EYFP and make.


From the figure 2, we learned with the increase of the expression intensity of makte, the specific valve of EYFP and mkate decreases. In another word, the circuit A’s output reaches saturation fast with the increase of copy number. We came to a conclusion circuit A’s adaptation to DNA copy number is higher than circuit B’s. So, the negative feedback works.

Parts

Discussion

Due to some restrictions in wetlab, we only finished the above-mentioned experiment. We found that the number of Hela cells who possesses high copy number is comparatively low. We also noticed the circuit C’s output is higher than expected in Figure 1.This may cause wrong judge when use the design to detect miR-21. Some measures will be taken to solve this question. Besides, we are going to endeavor to construct the other networks mentioned in modeling work.

Retrieved from "http://2013.igem.org/Team:Tsinghua-A/Wetlab"