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| − | <html>
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| − | <head>
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| − | <meta charset="utf-8">
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| − | <title>IGEM-PROJECT</title>
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| − | <style type="text/css">
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| − | #footer-box,#sponsor-box,.shadow3,#content,table,#catlinks {
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| − | background-color: transparent;
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| − | }
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| − | .mediawiki.ltr.ns-0.ns-subject.page-Team_HUST-China
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| − | #globalWrapper #top-section #p-logo a img {
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| − | visibility:hidden;
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| − | }
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| − | #p-logo {
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| − | background:url(https://static.igem.org/mediawiki/2014/5/52/Wiki_Logo_Transparent.png);
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| − | background-repeat: no-repeat;
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| − | }
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| − | #contentSub, #footer-box, #catlinks, #search-controls, #p-logo, .printfooter, .firstHeading, .visualClear{display: none;}
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| − | #globalWrapper, #content,#bodyContent { /*-- changes default wiki settings --*/
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| − | width: 100%;
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| − | height: 100%;
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| − | border: 0px;
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| − | background-color: transparent;
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| − | margin-top: -3px;
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| − | }
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| − | html, .wrapper { /*-- changes default wiki settings --*/
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| − | width: 100%;
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| − | height: 100%;
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| − | background-color: transparent;
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| − | }
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| − |
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| − | /* CSS Document */
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| − | *{
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| − | body{
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| − | font-family: Arial, Helvetica, Verdana;
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| − | color: #000;
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| − | font-size: 12px;
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| − | background-image: url(https://static.igem.org/mediawiki/2015/1/1c/Second_bd.jpg);
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| − | background-repeat: no-repeat;
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| − | background-attachment: fixed;
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| − | background-position: center top;
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| − | img{
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| − | ul ,li{
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| − | a{
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| − | html ,body{
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| − | height:100%;
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| − | }
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| − | .clearfix:after{content:".";display:block;height:0;clear:both;visibility:hidden;}
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| − | .clearfix{display:inline-table;}/* Hides from IE-mac \*/
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| − | *html .clearfix{height:1%;}
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| − | .clearfix{display:block;}/* End hide from IE-mac */
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| − | *+html .clearfix{min-height:1%;}
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| − | .top{
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| − | width:100%;
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| − | padding-top:43px;
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| − | height:388px;filter:alpha(opacity=50); opacity: 0.8;
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| − | }
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| − | .nav_bar{
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| − | background:#000;
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| − | opacity:0.95;
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| − | height:80px;
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| − | line-height:74px;
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| − | position:relative;
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| − | }
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| − | .nav{
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| − | width:1050px;
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| − | margin:0px auto;
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| − | position:relative;
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| − | height:80px;
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| − | line-height:4;
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| − | .nav li{
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| − | float:left;
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| − | display:block;
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| − | width:150px;
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| − | text-align:center;
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| − | position:relative;
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| − | }
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| − | .nav li a{
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| − | color:#fff;
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| − | font-size:16px;
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| − | display:block;
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| − | .nav li a:hover{
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| − | color:#C99091;
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| − | .sub{display:none;position:absolute;left:0px;top:80px;z-index:1;border:1px solid #666;border-top:0; background:#fff;}
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| − | .sub li{text-align:center;margin-bottom:-1px; display:block;width:148px; }
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| − | .sub li a{display:block; border-bottom:2px solid #666;padding:8px 0;height:28px;line-height:28px;color:#fff;}
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| − | .sub li a:hover{color:#000000;}
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| − | .logo{
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| − | width:150px;
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| − | margin:0px auto 0px;
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| − | text-align:center;
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| − | position:relative;
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| − | top:-20px;
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| − | height:122px;
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| − | .n1{
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| − | border-bottom:6px solid #2980b9;
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| − | .n5{
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| − | border-bottom:6px solid #0498f9;
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| − | .content_sub{
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| − | width:980px;
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| − | margin:0px auto;
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| − | padding:0px 10px;
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| − | text-align: justify;
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| − | }
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| − | .sc,.sc2{
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| − | width:980px;
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| − | margin:10px auto 60px;
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| − | clear:both;
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| − | .sc img{
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| − | margin:0px 10px 0px 20px;
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| − | }
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| − | .sc2 img{
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| − | float:left;
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| − | margin:0px 20px 0px 10px;
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| − | .content_sub h1{
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| − | font-size:30px;
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| − | margin-bottom:10px;
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| − | .content_sub p{
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| − | font-size:16px;
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| − | word-break:break-all;
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| − | }
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| − | .content_sub h2{
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| − | font-size:30px;
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| − | margin-bottom:10px;
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| − | }
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| − | #container{position:relative;width:766px;margin:0px auto; }
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| − | .templates li{float:left;height:160px;width:145px; margin:10px 10px; position:relative;left:0;top:0;z-index:0;}
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| − | .templates a{display:block;height:160px;position:relative;}
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| − | .templates img{width:145px;height:160px;}
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| − |
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| − | /* section */
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| − | .section{width:980px;margin:40px auto 0 auto;overflow:hidden;}
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| − | .section ul{width:996px;}
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| − | .section ul li{float:left;margin-right:14px;margin-bottom:13px;display:inline;width:318px;height:343px;overflow:hidden;position:relative;}
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| − | .section ul li .photo{width:318px;height:343px;overflow:hidden;}
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| − | .section .rsp{width:318px;height:343px;overflow:hidden;position: absolute;background:#000;top:0px;left:0px;}
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| − | .section .text{position:absolute;width:268px;height:303px;left:-318px;top:0px;overflow:hidden; font-size:16px;
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| − | color:#fff; padding:20px 25px;}
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| − | .section .text h3{width:318px;margin-top:130px;height:55px;line-height:55px;text-align:center;color:#FFFFFF;font-family:"microsoft yahei";font-size:26px;}
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| − | .footer{
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| − | margin:40px auto ;
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| − | text-align:center;
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| − | padding:20px 0px 0px;
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| − | } body {background-color: rgb(249,245,200);}
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| − | a.white:link {color:#FFF;} /* unvisited link */
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| − | a.white:visited {color:#FFF;} /* visited link */
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| − | a.white:hover {color:#FFF;} /* mouse over link */
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| − | a.white:active {color:#FFF;} /* selected link */
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| − | a{color:#909090}
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| − | .brightlink {color:#00BA00 !important;
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| − | }
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| − | .centertext {text-align: center;
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| − | }
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| − | a:visited{color:#909090}
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| − | p {border:none;
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| − | color:000000;
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| − | }
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| − | .teampagetext {vertical-align:text-top; display:inline; width:740px; float:left;}
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| − | h1 {border-color:white;
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| − | color:white;
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| − | }
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| − | h2 {color:white;}
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| − | h3 {margin-bottom:0px;
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| − | font-size:140%;
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| − | padding:0px;
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| − | color:white;}
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| − | #bd {
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| − | width:100%;
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| − | }
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| − | #bd #content{
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| − | width:967px;
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| − | margin:0 auto;
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| − | }
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| − | #bd #content .tp_zs{
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| − | width:100%;
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| − | margin:0 0 20px 0;
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| − | position:relative;
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| − | overflow:hidden;
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| − | }
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| − | #bd #content .tp_zs span{
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| − | margin-right: 10px;
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| − | display: block;
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| − | float: left;
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| − | cursor: pointer;
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| − | }
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| − | #bd #content .tp_zs span:hover img{
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| − | filter:alpha(opacity=50);
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| − | -moz-opacity:0.5;
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| − | -khtml-opacity: 0.5;
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| − | opacity: 0.5;
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| − | }
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| − | #bd #content .tp_zs .yb_tp{
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| − | margin-right:0px;
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| − | }
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| − | #bd #content .tp_zs span img{
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| − | width:234px;
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| − | }
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| − | #bd #content .wz_pb{
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| − | width:100%;
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| − | }
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| − | #bd #content .wz_pb .mainTitleHeader {
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| − | width: 937px;
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| − | float: left;
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| − | margin-top: 10px;
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| − | border-radius: 4px;
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| − | padding: 15px 15px 15px 15px;
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| − | font: sans-serif;
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| − | font-size: 13px;
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| − | color: rgba(225,225,225,.9);
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| − | text-align: justify;
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| − | background: rgba(168, 182, 120, .95);
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| − | }
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| − | #bd #content .wz_pb .mainTitleHeader p{
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| − | color: #212f20;
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| − | margin: 0px;
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| − | font-size: 140%;
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| − | font-weight: bold;
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| − | }
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| − | .mainContainer {
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| − | width: 97%;
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| − | float: left;
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| − | padding: 15px 15px 15px 15px;
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| − | font: sans-serif;
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| − | font-size: 13px;
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| − | color: #212f20;
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| − | text-align: justify;
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| − | }
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| − | .mainContainer .genpicfloatright {
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| − | border-radius: 4px;
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| − | margin-left: 20px;
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| − | float: right;
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| − | margin-bottom: 20px;
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| − | }
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| − | .mainContainer p {
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| − | color: #212f20;
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| − | font-size: 120%;
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| − | margin: .4em 0 .5em 0;
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| − | line-height: 1.5em;
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| − | }
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| − | .mainContainer .genpicfloatleft {
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| − | border-radius: 4px;
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| − | margin-right: 20px;
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| − | margin-bottom: 20px;
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| − | float: left;
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| − | }
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| − |
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| − |
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| − | </style>
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| − |
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| − | <script src="http://cdn.bootcss.com/jquery/1.11.3/jquery.min.js"></script>
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| − | <script type='text/javascript' src ='https://2015.igem.org/Team:Shiyan_SY_China/common/SuperSlide?action=raw&ctype=text/javascript'></script>
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| − | <script type='text/javascript' src ='https://2015.igem.org/Team:Shiyan_SY_China/common/public?action=raw&ctype=text/javascript'></script>
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| − |
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| − | </head>
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| − | <body>
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| − | <div class="top">
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| − | <div class="nav_bar">
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| − | <ul style="margin:0px auto;" class="nav clearfix">
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| − |
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| − | <li class="m n1">
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| − | <h3><a target="_blank" style="cursor: pointer;" onclick="location.href ='https://2015.igem.org/Team:Shiyan_SY_China';" title="">HOME</a></h3>
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| − | </li>
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| − |
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| − | <li class="m n2">
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| − | <h3><a target="_blank" style="cursor: pointer;" onclick="location.href ='https://2015.igem.org/Team:Shiyan_SY_China/Project.html';"title="">PROJECT</a></h3>
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| − | <ul style="margin:0px auto;" class="sub">
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| − | <li ><a style ="background:#27AE60" href="https://2015.igem.org/Team:Shiyan_SY_China/Project.html#t1">Description</a> </li>
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| − | <li ><a style ="background:#27AE60" href="https://2015.igem.org/Team:Shiyan_SY_China/Project.html#t2">Background</a> </li>
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| − | <li ><a style ="background:#27AE60" href="https://2015.igem.org/Team:Shiyan_SY_China/Project.html#t3">Design</a> </li>
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| − | <li ><a style ="background:#27AE60" href="https://2015.igem.org/Team:Shiyan_SY_China/Project.html#t4">Experiment&Steps</a> </li>
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| − | <li ><a style ="background:#27AE60" href="https://2015.igem.org/Team:Shiyan_SY_China/Project.html#t5">Results&Future</a> </li>
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| − | <li ><a style ="background:#27AE60" href="https://2015.igem.org/Team:Shiyan_SY_China/Project.html#t6">Parts</a> </li>
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| − | </ul>
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| − | </li>
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| − |
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| − | <li class="m n3">
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| − | <h3 style = "line-height: 2;"><a target="_blank" style="cursor: pointer;back" onclick="location.href ='https://2015.igem.org/Team:Shiyan_SY_China/Practice.html';" title="">HUMAN PRACTICES</a></h3>
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| − | <ul style="margin:0px auto;" class="sub">
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| − | <li ><a style ="background:#F39C12" href="https://2015.igem.org/Team:Shiyan_SY_China/Practice.html#t1">T-shirt</a> </li>
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| − | <li ><a style ="background:#F39C12" href="https://2015.igem.org/Team:Shiyan_SY_China/Practice.html#t2">Biology Speech</a> </li>
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| − | <li ><a style ="background:#F39C12" href="https://2015.igem.org/Team:Shiyan_SY_China/Practice.html#t3">IGEM Magazine</a> </li>
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| − | <li ><a style ="background:#F39C12" href="https://2015.igem.org/Team:Shiyan_SY_China/Practice.html#t4">Questionnaire</a> </li>
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| − | <li ><a style ="background:#F39C12" href="https://2015.igem.org/Team:Shiyan_SY_China/Practice.html#t5">Sina Weibo</a> </li>
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| − | </ul>
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| − | </li>
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| − | <li class="logo">
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| − | <img src="https://static.igem.org/mediawiki/2015/7/71/Heng_logo.png" width="122" height="122" style="cursor: pointer;" onclick="location.href ='http://www.igem.org/Main_Page';">
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| − | </li>
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| − | <li class="m n4">
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| − | <h3><a target="_blank" style="cursor: pointer;" onclick="location.href ='https://2015.igem.org/Team:Shiyan_SY_China/Safety.html';" title="">SAFETY</a></h3>
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| − | <ul style="margin:0px auto;" class="sub">
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| − | <li ><a style ="background:#BE382A" href="https://2015.igem.org/Team:Shiyan_SY_China/Safety.html#t1">Safety Design</a> </li>
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| − | <li ><a style ="background:#BE382A" href="https://2015.igem.org/Team:Shiyan_SY_China/Safety.html#t2">Lab security</a> </li>
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| − | <li ><a style ="background:#BE382A" href="https://2015.igem.org/Team:Shiyan_SY_China/Safety.html#t3">Safety Delivery</a> </li>
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| − | </ul>
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| − | </li>
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| − | <li class="m n5">
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| − | <h3><a target="_blank" style="cursor: pointer;" onclick="location.href ='https://2015.igem.org/Team:Shiyan_SY_China/Team.html';" title="">TEAM</a></h3>
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| − | <ul style="margin:0px auto;" class="sub">
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| − | <li ><a style ="background:#8E44AD" href="https://2015.igem.org/Team:Shiyan_SY_China/Team.html#t1">Members</a> </li>
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| − | <li ><a style ="background:#8E44AD" href="https://2015.igem.org/Team:Shiyan_SY_China/Team.html#t2">Instructors</a> </li>
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| − | <li ><a style ="background:#8E44AD" href="https://2015.igem.org/Team:Shiyan_SY_China/Team.html#t3">Attributions</a> </li>
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| − | <li ><a style ="background:#8E44AD" href="https://2015.igem.org/Team:Shiyan_SY_China/Team.html#t4">Collaborations</a> </li>
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| − | </ul>
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| − | </li>
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| − | <li class="m n6">
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| − | <h3><a target="_blank" style="cursor: pointer;" onclick="location.href ='https://2015.igem.org/Team:Shiyan_SY_China/Notebook.html';" title="">NOTEBOOK</a></h3>
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| − | <ul style="margin:0px auto;" class="sub">
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| − | <li ><a style ="background:#0498F9" href="https://2015.igem.org/Team:Shiyan_SY_China/Notebook.html#t1">Notebook</a> </li>
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| − |
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| − | </ul>
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| − | </li>
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| − | <br clear="all">
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| − | </ul>
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| − | </div>
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| − |
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| − |
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| − | <div>
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| − | <p style="width:800px; height:100px; padding:50px 80px; color:#000; text-align:center; margin:0 auto; font-size:48px; font-family:'Trebuchet MS', Helvetica, sans-serif;">You have to believe in yourself. That’s the secret of success. </p>
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| − | <p style="height:100px; color:#000; width:960px; line-height:100px; margin: 0 auto; text-align:right; font-size:24px; font-family:'Trebuchet MS', Helvetica, sans-serif;">
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| − | —— Charles Chaplin</p>
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| − | </div>
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| − | </div>
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| − | <div id="bd">
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| − | <div id="content">
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| − | <div class="wz_pb">
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| − | <div class="mainTitleHeader">
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| − | <a name="t3"><p style="font-weight: bold;">Design</p></a>
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| − | </div>
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| − | <div class="mainContainer">
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| − | <div>
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| − | <h style="font-size:15px;font-weight: bold;"> Experimental Design: </h></br>
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| − | <div> </div>
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| − | <h style="font-size:14px;font-weight: bold;"> 1.Inserted PARTS design: </h></br>
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| − | </div>
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| − | <div> </div>
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| − | <div align="center"><img src="https://static.igem.org/mediawiki/2015/b/b5/Project_001.jpg" width = "50%" > </div>
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| − | <p>
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| − | Note: F1:…,F2:…, F3:…, F4: ….</p>
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| − |
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| − | <div> </div>
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| − | <h style="font-size:14px;font-weight: bold;">2.Final Plasmid Design: </h></br>
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| − | <div> </div>
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| − | <div align="center"><img src="https://static.igem.org/mediawiki/2015/c/c8/Project_002.jpg" width = "50%" > </div>
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| − | <div> </div>
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| − | <h style="font-size:14px;font-weight: bold;">Experimental Procedure: </h></br>
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| − | <div> </div>
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| − | <div align="center"><img src="https://static.igem.org/mediawiki/2015/5/55/Project_003.jpg" width = "50%" > </div>
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| − | <div> </div>
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| − | <div>
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| − | <h style="font-size:14px;font-weight: bold;">
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| − | 1)Micro-organisms degrades organophosphorus, and organophosphorus-degradation enzyme opdA
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| − | </h>
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| − | <p>
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| − | Faced with the stress of human pollution such as pesticides, nature itself has evolved many methods to deal with these problems. For example, many natural micro-organisms contain enzymes to degrade organophosphorus pesticides. Currently the micro-organisms which are capable to degrade organophosphorus pesticides include bacteria, fungus, actinomycete and alga. As the research goes further, people find that these degrading effects come from secreting an enzyme, which can hydrolyze phosphoester bonds, organophosphorus degradation enzyme. Because each organophosphorus pesticide has similar structure and protein sequence, one kind of organophosphorus degradation enzyme is capable todegrade multiple kinds of organophosphorus pesticides. Organophosphorus-degradation enzyme has been mostly recognized as the best method to eliminate pesticide residues currently. At present, many enzymes have been identified to be used to degrade organophosphate pesticides. Among these enzymes, the organophosphorus-degradation enzyme (opdA) which comes from Agrobacterium radiobacter P230 has wider targets and higher enzyme-catalyst efficiency. In recent years, the research on the structure and function of organophosphorus-degradation enzyme has gained promising progress,. Thus, it is possible to improve the properties of organophosphorus-degradation enzyme through genetic engineering and protein engineering method, which meet requirements of different applications.
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| − | </p>
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| − |
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| − | </div>
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| − | <div>
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| − | <h style="font-size:14px;font-weight: bold;">
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| − | 2)In this project, we will use organophosphorus-degradation enzyme opdA to eliminate residual organophosphorus pesticides on fruits and vegetables.
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| − | </h>
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| − | <p>
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| − | The organophosphorus-degradation enzyme (opdA) gene opdA （NCBI genbank：Accession: AY043245.2） programmed by Agrobacterium radiobacter contains 1,155 nucleic acids, programming 384 amino acid residues. The N-terminal of protein sequence is the signal peptide while the C-terminal is the degradation-enzyme sequence. The nucleic acid sequence and amino acid sequence are as follows:
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| − | </p>
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| − | <h style="font-size:14px;font-weight: bold;">
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| − | Nucleotide sequence
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| − | </h>
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| − | <p>
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| − | at gcaaacgaga agagatgcac ttaagtctgc ggccgcaata actctgctcg gcggcttggc tgggtgtgca agcatggccc gaccaatcgg tacaggcgat ctgattaata ctgttcgcgg ccccattcca gtttcggaag cgggcttcac actgacccat gagcatatct gcggcagttc ggcgggattc ctacgtgcgt ggccggagtt tttcggtagc cgcaaagctc tagcggaaaa ggctgtgaga ggattacgcc atgccagatc ggctggcgtg caaaccatcg tcgatgtgtc gactttcgat atcggtcgtg acgtccgttt attggccgaa gtttcgcggg ccgccgacgt gcatatcgtg gcggcgactg gcttatggtt cgacccgcca ctttcaatgc gaatgcgcag cgtcgaagaa ctgacccagt tcttcctgcg tgaaatccaa catggcatcg aagacaccgg tattagggcg ggcattatca aggtcgcgac cacagggaag gcgaccccct ttcaagagtt ggtgttaaag gcagccgcgc gggccagctt ggccaccggt gttccggtaa ccactcacac gtcagcaagt cagcgcgatg gcgagcagca ggcagccata tttgaatccg aaggtttgag cccctcacgg gtttgtatcg gtcacagcga tgatactgac gatttgagct acctaaccgg cctcgctgcg cgcggatacc tcgtcggttt agatcgcatg ccgtacagtg cgattggtct agaaggcaat gcgagtgcat tagcgctctt tggtactcgg tcgtggcaaa caagggctct cttgatcaag gcgctcatcg accgaggcta caaggatcga atcctcgtct cccatgactg gctgttcggg ttttcgagct atgtcacgaa catcatggac gtaatggatc gcataaaccc agatggaatg gccttcgtcc ctctgagagt gatcccattc ctacgagaga agggcgtccc gccggaaacg ctagcaggcg taaccgtggc caatcccgcg cggttcttgt caccgaccgt gcgggccgtc gtgacacgat ctgaaacttc ccgccctgcc gcgcctattc cccgtcaaga taccgaacga tga
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| − | </p>
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| − | <h style="font-size:14px;font-weight: bold;">
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| − | Amino acid sequence
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| − | </h>
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| − | <p style = "word-break:break-all;">
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| − | MQTRRDALKSAAAITLLGGLAGCASMARPIGTGDLINTVRGPIPVSEAGFTLTHEHICGSSAGFLRAWPEFFGSRKALAEKAVRGLRHARSAGVQTIVDVSTFDIGRDVRLLAEVSRAADVHIVAATGLWFDPPLSMRMRSVEELTQFFLREIQHGIEDTGIRAGIIKVATTGKATPFQELVLKAAARASLATGVPVTTHTSASQRDGEQQAAIFESEGLSPSRVCIGHSDDTDDLSYLTGLAARGYLVGLDRMPYSAIGLEGNASALALFGTRSWQTRALLIKALIDRGYKDRILVSHDWLFGFSSYVTNIMDVMDRINPDGMAFVPLRVIPFLREKGVPPETLAGVTVANPARFLSPTVRAVVTRSETSRPAAPIPRQDTER
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| − | </p>
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| − | <h style="font-size:14px;font-weight: bold;">
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| − | DNA SEQUENCE
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| − | </h>
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| − | <p>
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| − | at gcaaacgaga agagatgcac ttaagtctgc ggccgcaata actctgctcg gcggcttggc tgggtgtgca agcatggccc gaccaatcgg tacaggcgat ctgattaata ctgttcgcgg ccccattcca gtttcggaag cgggcttcac actgacccat gagcatatct gcggcagttc ggcgggattc ctacgtgcgt ggccggagtt tttcggtagc cgcaaagctc tagcggaaaa ggctgtgaga ggattacgcc atgccagatc ggctggcgtg caaaccatcg tcgatgtgtc gactttcgat atcggtcgtg acgtccgttt attggccgaa gtttcgcggg ccgccgacgt gcatatcgtg gcggcgactg gcttatggtt cgacccgcca ctttcaatgc gaatgcgcag cgtcgaagaa ctgacccagt tcttcctgcg tgaaatccaa catggcatcg aagacaccgg tattagggcg ggcattatca aggtcgcgac cacagggaag gcgaccccct ttcaagagtt ggtgttaaag gcagccgcgc gggccagctt ggccaccggt gttccggtaa ccactcacac gtcagcaagt cagcgcgatg gcgagcagca ggcagccata tttgaatccg aaggtttgag cccctcacgg gtttgtatcg gtcacagcga tgatactgac gatttgagct acctaaccgg cctcgctgcg cgcggatacc tcgtcggttt agatcgcatg ccgtacagtg cgattggtct agaaggcaat gcgagtgcat tagcgctctt tggtactcgg tcgtggcaaa caagggctct cttgatcaag hgcgctcatcg accgaggcta caaggatcga atcctcgtct cccatgactg gctgttcggg ttttcgagct atgtcacgaa catcatggac gtaatggatc gcataaaccc agatggaatg gccttcgtcc ctctgagagt gatcccattc ctacgagaga agggcgtccc gccggaaacg ctagcaggcg taaccgtggc caatcccgcg cggttcttgt caccgaccgt gcgggccgtc gtgacacgat ctgaaacttc ccgccctgcc gcgcctattc cccgtcaaga taccgaacga tga
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| − | </p>
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| − | <h style="font-size:14px;font-weight: bold;">
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| − | PROTEIN SEQUENCE
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| − | </h>
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| − | <p style = "word-break:break-all;">
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| − | MQTRRDALKSAAAITLLGGLAGCASMARPIGTGDLINTVRGPIPVSEAGFTLTHEHICGSSAGFLRAWPEFFGSRKALAEKAVRGLRHARSAGVQTIVDVSTFDIGRDVRLLAEVSRAADVHIVAATGLWFDPPLSMRMRSVEELTQFFLREIQHGIEDTGIRAGIIKVATTGKATPFQELVLKAAARASLATGVPVTTHTSASQRDGEQQAAIFESEGLSPSRVCIGHSDDTDDLSYLTGLAARGYLVGLDRMPYSAIGLEGNASALALFGTRSWQTRALLIKALIDRGYKDRILVSHDWLFGFSSYVTNIMDVMDRINPDGMAFVPLRVIPFLREKGVPPETLAGVTVANPARFLSPTVRAVVTRSETSRPAAPIPRQDTER
| |
| − | </p>
| |
| − | </div>
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| − | <div>
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| − | <h style="font-size:14px;font-weight: bold;">
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| − | 3)Genetically engineered E.Colibacteria
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| − | </h>
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| − | <p>
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| − | In this project, we will use E. Coli to construct genetically engineered bacteria which can secrete organophosphorus-degradation enzyme opdA protein, to eliminate the pesticides.
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| − | </p>
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| − | <p>
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| − | Genetically engineered bacteria are bacteria which can channel target gene into bacteria to express the genes and produce required protein.
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| − | </p>
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| − | <p>
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| − | Currently, the mostly used genetically engineered bacteria all over the world are still E. Coli. E. Coli have explicit genetic background, fast growth rate, limited antibiotics resistance, Thus, E. Coli, are easy to be used in any production magnitude from laboratory to industry production. (For example: scientists introduced human insulin gene into E. Coli genome. E. Coli can express functional human insulin protein, which is used as a medicine for diabetes treatment. Human insulin production from E. coli has been applied to industry and this method is also widely used in biotech industry for other drug purposes In 1981, human insulin gene products were put into market and solved the problem of lack of insulin sources).
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| − | </p>
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| − | </div>
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| − | <div>
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| − | <h style="font-size:14px;font-weight: bold;">
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| − | 4）RNA Thermometer
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| − | </h>
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| − | <p>
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| − | An RNA thermometer (or RNA thermosensor) is a temperature-sensitive non-coding RNA molecule which regulates gene expression. RNA thermometers often regulate genes required during either a heat shock or cold shock response. In general, RNA thermometers operate by changing their secondary structure in response to temperature fluctuations. This structural transition can then expose or occlude important regions of RNA such as a ribosome binding site, which then affects the translation rate of a nearby protein-coding gene.
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| − | </p>
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| − | <div> </div>
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| − | <p>
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| − | Below is a schematic diagram of Thermometer RNA:
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| − | </p>
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| − | <div> </div>
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| − | <div align="center"><img src="https://static.igem.org/mediawiki/2015/c/c7/Project_004.jpg" width = "20%" > </div>
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| − | <div> </div>
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| − | <p>
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| − | In this project, the Thermometer RNA we use has a sensitive temperature of 32℃, which means in environment above 32C, RNA translation process can be started. Its DNA sequence is as follows:
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| − | </p>
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| − | <p style = "word-break:break-all;">
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| − | Ccgggcgcccttcgggggcccggcggagacgggcgccggaggtgtccgacgcctgctcgtccagtctttgctcagtggaggattactag
| |
| − | </p>
| |
| − | </div>
| |
| − | <div>
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| − | <h style="font-size:14px;font-weight: bold;">
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| − | 5)ompA signal peptide
| |
| − | </h>
| |
| − | <p>
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| − | In this project, to secrete the opdA enzyme from inside the bacteria to outside, an ompA signal peptide is introduced into the construction. By adding this secreting peptide, OpdA is secreted outside the E. Coli, without its accumulating inside.
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| − | </p>
| |
| − | <p>
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| − | Signal peptide often refers to a N-terminal amino acid sequence which is used to direct the trans-membrane process in newly synthesized proteins. There is a segment of RNA area programming hydrophobic amino acid sequence which is generally behind the start codon. This amino acid sequence is called signal peptide sequence, which introduces protein into sub-cellular organelles which contain different membrane structures. If the function of this signal peptide is to secrete protein outside the cell walls, this signal peptide is called secretion signal peptide.
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| − | </p>
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| − | <p>
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| − | As we know, on natural conditions, ompA which exists inside Agrobacterium radiobacter bacteria has its own signal peptide. However, this original signal peptide is only used by Agrobacterium radiobacter and is not necessarily functional inE. Coli. Therefore, we need to select an E. colisignal peptides to achieve our plan.
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| − | </p>
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| − | <p>
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| − | The ompA signal peptide used in this project is one of the E. coli secreting signal peptides and can lead secretion of its downstream protein outside of its host E. coli. The DNA sequence of this signal peptide is as follows:
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| − | </p>
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| − | <p style = "word-break:break-all;">
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| − | Atgaaaaaaaccgctatcgcgatcgcagttgcactggctggtttcgctaccgttgcgcaggcc
| |
| − | </p>
| |
| − | </div>
| |
| − | <div>
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| − | <h style="font-size:14px;font-weight: bold;">
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| − | 6)Suicide gene ccdb
| |
| − | </h>
| |
| − | <p>
| |
| − | In this project, we consider not only how to use genetically engineered bacteria to eliminate organophosphorus, but also after organophosphorus is eliminated, how we should deal with the rest genetically engineered bacteria. Genetically engineered bacteria, after all, come from E. coli. We don’t want them released into environment or remaining on the surface of fruits and vegetables. Thus we must design a method to eliminate them. Consequently, we decide to design a suicide gene into our genetically engineered bacteria, which is inactivated under normal condition and only activated under special environments to eliminate the bacteria and avoid “secondary pollution”.
| |
| − | </p>
| |
| − | <p>
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| − | To achieve the above goal, we select suicide gene ccdb. ccdB is a known toxin system, which exists in pathogenic E. coli F plasmid. The ccdB gene programs a toxin protein CcdB. On conditions where there is a lack of antitoxin, CcdB poisons gyrase inside cells to interfere with the DNAsynthesis and damage host cells. The ccdb sequence we use in this project is as follows:
| |
| − | </p>
| |
| − | <p style = "word-break:break-all;">
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| − | Atgcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattgacacgcccgggcgacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaactttacccggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgccggtctccgttatcggggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatataa
| |
| − | </p>
| |
| − | </div>
| |
| − | <div>
| |
| − | <h style="font-size:14px;font-weight: bold;">
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| − | 7)RecA(SOS) promoter
| |
| − | </h>
| |
| − | <p>
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| − | In the project we chose a RecA(SOS) promoter to control to suicide gene ccdB to express. When the bacteria receive ultraviolet lights, DNA injurｙ activate the RecA repairing system. The system can activate RecA(SOS) promoter to drive the downstream ccdB suicide gene’s transcription. In this way, we can control our engineering bacteria by eliminating them with ultraviolet rays to avoid secondary pollution.
| |
| − | </p>
| |
| − | <p>
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| − | The ccdb sequence we use in this project is as follows:
| |
| − | </p>
| |
| − | <p style = "word-break:break-all;">
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| − | Aacaatttctacaaaacacttgatactgtatgagcatacagtataattgcttcaacagaacatattgactatccggtattacccggcatgacaggagtaaaaatggctatcgacgaaaacaaacagaaagcgttggcggcagcactgggccagattgagaaacaatttggtaaaggctccatcatgtaataa
| |
| − | </p>
| |
| − | </div>
| |
| − | <div>
| |
| − | <h style="font-size:14px;font-weight: bold;">
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| − | 8)The design of the biobricks in this project referred to a lot of information from other igem teams in past years and from other scientific projects in the society. The information was coordinated and integrated in this project, and formed the research design and contents of this project.
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| − | </h>
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| − | <p>
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| − | (1)The strong initiation sequence used in this project referred to BB_J23106 from igem
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| − | </p>
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| − | <p>
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| − | (2)The RNA thermometer sequence used in this project referred to BB_K115017 from igem
| |
| − | </p>
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| − | <p>
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| − | (3)The organophosphorus-degradation enzyme opdA gene sequence used in this project referred to BB_K21509, BB_K215091 and BB_K1010008 from igem
| |
| − | </p>
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| − | <p>
| |
| − | (4)The repair enzyme induced promoter RecA (SOS) sequence used in this project referred to BB_J22106 from igem
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| − | </p>
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| − | <p>
| |
| − | (5)The suicide gene ccdB sequence used in this project referred to BB_K145151 and BB_K1010007 from igem
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| − | </p>
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| − | <p>
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| − | (6)The ompA signal peptide sequence used in this project referred to sequence with the code AJ617284.1 from GenBank
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| − | </p>
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| − |
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| − | </div>
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| − | </div>
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| − | </div>
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| − | <div> </div>
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| − | <div class="footer">
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| − | <p>© 2015 Shiyan_SY_China iGEM Team. All rights reserved.
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| − | </p>
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