Team:Gothenburg-Sweden/test

From 2010.igem.org

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(119 intermediate revisions not shown)
Line 1: Line 1:
<html xmlns="http://www.w3.org/1999/xhtml">
<html xmlns="http://www.w3.org/1999/xhtml">
 +
<head>
 +
<meta http-equiv="content-type" content="text/html; charset=utf-8" />
 +
<title>Chalmers University of Technology</title>
<style type="text/css">
<style type="text/css">
-
/* Wiki Hacks - START */
+
 
-
/* Author: Pieter van Boheemen */
+
/* wiki modificiations start */
-
/* Team: TU Delft */
+
-
#globalWrapper { background-color: transparent; border: none; margin: 0; padding: 0;}
+
-
#content { background-color: transparent; border: none; padding: 0; margin: 0; width: 100%;}
+
-
#bodyContent { border: none; padding:0; margin:0; width:100%;}
+
-
#top-section { height: 20px; margin-top: -5px; margin-left: auto; margin-right: auto; margin-bottom: 0 !important; margin-bottom: 10px; padding:0; border: none;}
+
-
#p-logo { height:1px; overflow:hidden; display: none;}
+
-
#search-controls { overflow:hidden; display:block; background: none; position: absolute; top: 100px; right: 40px;}
+
-
.left-menu { width: 400px; display:block; float:left; margin-top:-80px; border: none;}
+
-
.left-menu ul { border: none; }
+
-
#menubar.right-menu { width:300px; display:block; float:left; margin-top:-80px; border: none;}
+
-
.right-menu ul { border: none;}
+
-
#footer-box { background-color: transparent; border: none; width: 965px; margin: 0 auto; padding: 0;}
+
-
#footer { border: none; width: 965px; margin: 0; padding: 0;}
+
-
.firstHeading { display: none;}
+
-
#f-list a { color: #333; font-size: 10px;}
+
-
#f-list a:hover { color: #666;}
+
-
#footer ul { margin: 0; padding: 0;}
+
-
#footer ul li { margin-top: 0; margin-bottom: 0; margin-left: 10px; margin-right: 10px; padding: 0;}
+
-
/* Wiki Hacks - END */
+
#contentSub {
#contentSub {
display:none;
display:none;
}
}
 +
#siteSub {
#siteSub {
-
display:none;
+
display:none;padding:-200px;margin:0;
}
}
-
#search-controls {
 
-
display:none;
 
-
}
 
.firstHeading {
.firstHeading {
-
display:none;
+
display:none;padding:0;margin:0;
}
}
#search-controls {
#search-controls {
-
margin-top:30px;
+
padding-top:10px;
}
}
-
#footer-box {
+
 
-
display:none;
+
 
 +
form {
 +
margin: 0px;
 +
padding: 0px;
}
}
 +
 +
h2 {background:transparent; text-decoration:none;  }
 +
#globalWrapper { background-color: transparent; border: none; margin-bottom: 0px; padding: 0;}
 +
#content { background-color: transparent; border: none; padding: 0; margin-bottom: 0px; width: 100%;}
 +
#bodyContent { border: none; padding:0; margin-bottom:0px; width:100%;}
 +
 +
#top-section { background-color: transparent; height: 20px; margin-top: -30px; margin-left: auto; margin-right: auto; margin-bottom: 0 !important; margin-bottom: 0; padding:0; border: none; }
 +
#p-logo {  display: none;}
 +
#search-controls { display:none;}
 +
 +
#menubar.left-menu {width: 400px;  display:block; float:left; margin-top:-50px; border: none;}
 +
#menubar.left-menu ul { border: none;}
 +
#menubar.left-menu a{color:#5698b0;}
 +
#menubar.left-menu a:hover{color:#e6f6fb;}
 +
#menubar.left-menu a:selected{color:#e6f6fb;}
 +
 +
#menubar.right-menu {  width:300px; display:block; float:left; margin-top:-50px; border: none;}
 +
#menubar.right-menu ul {  border: none;}
 +
#menubar.right-menu a{color:#5698b0;}
 +
#menubar.right-menu a:hover{color:#e6f6fb;}
 +
 +
 +
/* wiki modifications end/
 +
 +
 +
 +
 +
/*to be modified */
 +
 +
body {background:url(https://static.igem.org/mediawiki/2010/8/8e/Shadow.png) repeat-y top center;
 +
  padding: 0 0 0 0;
 +
 
 +
    }
a{
a{
     font-family: Arial, Helvetica, sans-serif;
     font-family: Arial, Helvetica, sans-serif;
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     color: #06639e;
     color: #06639e;
     text-decoration:none;
     text-decoration:none;
 +
    padding:0;
 +
    margin:0;
}
}
 +
a:hover{
a:hover{
 +
padding:0;margin:0;
     text-decoration: underline;
     text-decoration: underline;
     color: #000066;
     color: #000066;
 +
}
}
 +
 +
a:visited{color:#06639E;}
*
*
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}
}
-
body
 
-
{
 
-
    font: 12px Arial, Helvetica, sans-serif;
 
-
    color: #000000;
 
-
    line-height: 20px;
 
-
    background: #FEFEFE url(https://static.igem.org/mediawiki/2010/5/5b/BgGo.jpg);
 
-
    background-repeat:repeat-x;
 
-
    background-position: top;
 
-
}
 
-
#mainbg{
+
#all{
-
    width: 1016px;
+
  padding:0;
-
    margin: 0 auto;
+
  margin:0;
-
    padding-top:20px;
+
  padding-left:10%;
-
}
+
  background:  url(https://static.igem.org/mediawiki/2010/3/3a/Bg.png) top center repeat-y;
 +
}  
-
#main{
 
-
    margin: 0 auto;
 
-
    width: 1016px;
 
-
    padding-top: 30px;
 
-
}
 
#header{
#header{
-
    height: 300px;
+
  height: 290px;
-
    width: 100%;
+
  background:url(https://static.igem.org/mediawiki/2010/5/50/BannerGo.png) no-repeat;  
-
padding-left:85px;
+
  margin-left:15px;
-
}
+
 +
}  
-
 
+
#search {height: 30px;}
-
#content{
+
#myContent{
 +
margin-top: 0px;
}
}
 +
 +
/* left layer starts */
#left{
#left{
-
     width:180px;
+
     width:138px;
-
 
+
    padding-left:45px;
     float: left;
     float: left;
-
 
 
}
}
-
#left h2 {
+
.Title1 {
     font-family: Comic Sans;
     font-family: Comic Sans;
     font-weight:600;
     font-weight:600;
-
     color: #ffffff;
+
     color: #fff;
     font-size: 16px;
     font-size: 16px;
     text-align:center;
     text-align:center;
 +
    width:100%;
     height: 30px;
     height: 30px;
-
     padding-top: 9px;
+
     padding-top: 7px;
-
     background: url(https://static.igem.org/mediawiki/2010/6/60/Title_left.png);
+
    margin-top:7px;
 +
     background: url(https://static.igem.org/mediawiki/2010/a/aa/CatTitle.png);
     background-repeat:no-repeat;
     background-repeat:no-repeat;
     background-position:top left;
     background-position:top left;
}
}
-
#left .company{
+
.left_grad{
-
     padding-top: 10px;
+
   
-
font-size:11px;
+
    background-position: top left;
 +
     padding-top: 0px;
 +
    margin-top: 0px;
}
}
-
.numleft{
+
.categories{
-
     background: url(https://static.igem.org/mediawiki/2010/0/0a/Numbg.png);
+
   
-
    background-repeat: no-repeat;
+
     background: url(https://static.igem.org/mediawiki/2010/1/10/CatCon.png) top repeat-y;
-
    background-position: left top;
+
     min-height:100px;
-
    float:left;
+
     border: 1px solid #0485B0;
-
    width: 37px;
+
-
     min-height: 37px;
+
-
     font-family: Arial,Helvetica,sans-serif;
+
-
    font-size: 16px;
+
-
    font-weight: bold;
+
-
    color: #ffffff;
+
-
    text-align: center;
+
-
    margin: 0px 8px 0px 8px;
+
}
}
 +
#news {
 +
  padding-top:0px;
 +
  background: url(https://static.igem.org/mediawiki/2010/1/10/CatCon.png) left top repeat-y;
 +
  min-height:100px;
 +
  border: 1px dotted #0485B0; 
 +
 +
}
 +
#news p{
 +
    color:#07374E;
 +
    font-weight:normal;
 +
    font-size: 11px;
 +
text-align:left;
 +
    text-decoration:none;
 +
   
 +
    padding: 10px 5px 0px 15px;
 +
 +
   
 +
display:block;
 +
}
-
.numleft p{
+
#news a:hover {
-
    padding-top: 10px;
+
border-bottom: 1px dotted #06639e;
}
}
-
 
+
.categories a{
-
.newsright{
+
     font-family: Arial, Helvetica, sans-serif;
-
     padding-left: 53px;
+
-
    padding-right: 10px;
+
-
 
+
-
font-family: Arial, Helvetica, sans-serif;
+
     font-weight:normal;
     font-weight:normal;
-
     color: #062F42;
+
     color: #0485B0;
     text-decoration: none;
     text-decoration: none;
-
font-size:11px;
+
    text-align:left;
 +
    padding: 4px 0px 4px 20px;
 +
    font-size:12px;
 +
    padding-top:5px;
 +
    display:block;
 +
 
}
}
-
#left .read a{
+
.categories a:hover{
-
    color: #06639e;
+
  text-decoration:none;
-
    font-weight:normal;
+
-
    font-style: italic;
+
-
    font-size: 12px;
+
-
    text-decoration:none;
+
-
    border-bottom: 1px #06639e;
+
-
    margin-right: 10px;
+
-
}
+
-
#left .read a:hover{border-bottom: 1px dotted #06639e;}
+
  font-size:12px;
-
 
+
  font-weight:700;
-
.left_grad{
+
  color: #fff;
-
    background: url(https://2010.igem.org/Image:Left_grad.png) repeat-y;
+
  background:url(https://static.igem.org/mediawiki/2010/4/47/CatAhover.png);
-
    background-position: top left;
+
-
+
}
}
 +
/* left layer ends */
-
.list_border{
+
/* center layer starts */
-
    background: url(https://static.igem.org/mediawiki/2010/d/d0/Left_grad.png) repeat-y;
+
-
    background-position:left top;
+
-
    height: 5px;
+
-
    margin-bottom: 10px;
+
-
}
+
#center{
#center{
-
     width: 616px;
+
     width: 600px;
-
     padding-left: 10px;
+
     padding-left: 20px;
     float: left;
     float: left;
}
}
-
 
#center p{
#center p{
     padding-left:0px;
     padding-left:0px;
     padding-right:0px;
     padding-right:0px;
     padding-bottom: 10px;
     padding-bottom: 10px;
-
color: #07374E;
+
 +
}
 +
    /* menu starts */
 +
 +
     
 +
        #Title2{
 +
 
 +
          height: 40px;
 +
          width: 600px;
 +
       
 +
          background: url(https://static.igem.org/mediawiki/2010/5/50/Title_center1.png);
 +
          background-repeat:no-repeat;
 +
          background-position: center;
 +
          }
 +
 +
#sddm
 +
{
 +
 
 +
  padding-left:62px;
 +
  padding-top: 0px;
 +
}
 +
 +
#sddm li
 +
{     
 +
list-style: none;
 +
float:left;
 +
text-align:center;
 +
}
 +
 +
 +
#sddm li a
 +
 +
{ display: block;
 +
padding: 0px 0px;
 +
width: 96px;
 +
height:33px;
 +
text-align: center;
 +
text-decoration: none;
 +
font-size:  15px;
 +
font-family: Rockwell;
 +
font-weight:normal;
 +
padding-top:10px;
 +
                       
 +
}
 +
#active {
 +
           
 +
}
 +
#active a{
 +
 +
background: url(https://static.igem.org/mediawiki/2010/0/0c/MActive2.png) top center repeat-y;
 +
        color:#FFFFFF;
 +
font:  13px arial !important;
 +
font-weight:600 !important;
 +
}
 +
#sddm li a:hover
 +
{ background: url(https://static.igem.org/mediawiki/2010/0/0c/MActive2.png) top center repeat-y;
 +
color:#fff;
 +
font:  13px arial;
 +
font-weight:600;
 +
}
 +
 +
 +
#sddm div
 +
{ position: absolute;
 +
visibility: hidden;
 +
margin-top: -5px;
 +
                     
 +
padding: 0px;
 +
background: #fff ;
 +
border: 1px solid #9CD2ED ;
 +
}
 +
 +
 +
#sddm div a
 +
{ position: relative;
 +
height:16px;
 +
display: block;
 +
margin: 0px;
 +
padding: 4px 20px;
 +
                                padding-top:auto;
 +
                                padding-bottom:auto;
 +
width:auto;
 +
min-width: 100px;
 +
font-family: sans-serif, Tahoma, Verdana, Geneva, Arial, Helvetica;
 +
text-align: left;
 +
text-decoration: none;
 +
background: transparent;
 +
color: #2e6d95;
 +
font-size: 11px ;
 +
border:none;
 +
 +
}
 +
 +
#sddm div a:hover
 +
{ background: #61b5da  url(https://static.igem.org/mediawiki/2010/9/9d/MArrow.png) no-repeat;
 +
 +
color: #FFF;
 +
text-decoration:none;
 +
font:  11px arial;
 +
font-weight:500;
 +
}
 +
 +
/* menu ends */
 +
 +
#text{
 +
    text-align:left;
 +
width:100%;
 +
margin: 0px 10px 10px 20px;
 +
    background: #fff ;
}
}
 +
#text p
 +
{
 +
  padding: 10px 10px 10px 25px;
 +
  font-family: "Times New Roman";
 +
  font-size:13px;
 +
  line-height:19px;
 +
}
-
.text{
+
.logo{
-
    padding: 10px;
+
float:left;
-
    background: #ffffff url(https://static.igem.org/mediawiki/2010/d/da/Text_bg.png);
+
padding: 10px 20px 10px 20px;
-
     background-position: bottom;
+
}
-
     background-repeat: repeat-x;
+
 
-
    min-height: 64px;
+
#text p{color: #07374E;}
-
+
 
 +
.read{
 +
     text-align: right;
 +
     padding-bottom:5px;
 +
  /* padding-right: 5px;*/
}
}
-
 
+
#myTitle{
-
#center H1{
+
     font-family: Arial, Helvetica, sans-serif;
     font-family: Arial, Helvetica, sans-serif;
     font-weight:normal;
     font-weight:normal;
Line 218: Line 353:
     font-size: 16px;
     font-size: 16px;
     height: 36px;
     height: 36px;
-
     width: 616px;
+
      
     padding-top: 10px;
     padding-top: 10px;
-
text-align:center;
+
    padding-right:-200px;
-
     background: url(https://static.igem.org/mediawiki/2010/6/64/Title_center.png);
+
    margin-left: 20px;
 +
    text-align:center;
 +
     background: url(https://static.igem.org/mediawiki/2010/6/64/Title_center.png) left top;
     background-repeat:no-repeat;
     background-repeat:no-repeat;
-
    background-position: top;
+
 
}
}
-
.read{
+
 
-
    text-align: right;
+
#team {
-
    padding-bottom:5px;
+
padding: 20px 10px 20px 30px;
-
    padding-right: 5px;
+
 
}
}
-
#center .read{
+
/* center ends */
-
     height: 26px;
+
 
-
     padding-top: 3px;
+
/* right starts */
-
     background: url(https://static.igem.org/mediawiki/2010/0/07/Read.png);
+
 
-
    background-repeat: no-repeat;
+
#right{
-
     background-position: top;
+
     width:160px;
 +
     padding-left: 20px;
 +
     /*padding-right: 135px;*/
 +
     float:left;
}
}
-
#center .read a{
+
#right .read a{
-
     color: #ffffff;
+
   
 +
     color: #06639e;
     font-weight:normal;
     font-weight:normal;
     font-style: italic;
     font-style: italic;
     font-size: 12px;
     font-size: 12px;
-
    padding-right: 15px;
 
     text-decoration:none;
     text-decoration:none;
-
 
+
    border-bottom: 1px #06639e;
 +
    padding-right:10px;
}
}
-
.read a:hover{
+
#right .read a:hover{border-bottom: 1px dotted #06639e;}
-
    text-decoration:underline;
+
-
}
+
-
#right{
 
-
    width:180px;
 
-
    padding-right: 19px;
 
-
    float:right;
 
-
}
 
-
#right h2 {
+
#lab{
 +
 +
padding-top:0px;
 +
background: url(https://static.igem.org/mediawiki/2010/1/10/CatCon.png) top left repeat-y;
 +
min-height:100px;
 +
/*margin-left:30px;*/
 +
border: 1px dotted #0485B0;
 +
}
 +
.numleft{
 +
background: url(https://static.igem.org/mediawiki/2010/2/22/Numbg1.png);
 +
background-repeat: no-repeat;
 +
background-position: left top;
 +
float:right;
 +
width: 37px;
 +
min-height: 37px;
 +
font-family: Arial,Helvetica,sans-serif;
 +
 +
font-weight: bold;
 +
color: #ffffff;
 +
text-align: center;
 +
margin: 25px 8px 0px 8px;
 +
}
 +
 +
 +
.numleft p{
 +
padding-top: 2px;
 +
                          padding-left: 1px;
 +
font-size: 16px;
 +
 +
}
 +
 +
 +
.newsright{
 +
    text-align:left;
 +
margin: 10px 50px 0 10px;
 +
}
 +
 +
.newsright p{
 +
font-size:11px;
 +
            color:#07374E;
 +
            margin-top:7px;
 +
 +
}
 +
 
 +
.Title3 {
     font-family: Comic Sans;
     font-family: Comic Sans;
     font-weight:600;
     font-weight:600;
-
     color: #ffffff;
+
     color: #fff;
     font-size: 16px;
     font-size: 16px;
     text-align:center;
     text-align:center;
 +
    width:100%;
     height: 30px;
     height: 30px;
-
    padding-top: 9px;
+
    padding-top: 7px;
-
     background: url(https://static.igem.org/mediawiki/2010/6/60/Title_left.png);
+
    margin-top:7px;
 +
margin-left:0px;
 +
     background: url(https://static.igem.org/mediawiki/2010/c/cf/CatTitle1.png) ;
     background-repeat:no-repeat;
     background-repeat:no-repeat;
     background-position:top left;
     background-position:top left;
}
}
-
.categories{
 
-
    min-height:100px;
 
-
}
 
-
.categories a{
+
/* right ends */
-
  font-family: Arial, Helvetica, sans-serif;
+
-
    font-weight:normal;
+
-
    color: #07374E;
+
-
    text-decoration: none;
+
-
font-size:11px;
+
-
}
+
-
.categories a:hover{
 
-
    background-color:#2286C9;
 
-
color:#FFFFFF;
 
-
}
 
-
 
-
.categories ul{
 
-
    padding-left: 5px;
 
-
    padding-right: 5px;
 
-
}
 
-
 
-
.categories ul li{
 
-
    list-style: none;
 
-
    padding: 3px 5px 3px 5px;
 
-
    border-bottom: 1px dotted #2286C9;
 
-
}
 
 +
/* footer starts */
-
#footer
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+
                         
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 +
                             
 +
                              <div class="Title1">Team</div>
 +
                              <div class="left_grad">
 +
                                  <div class="categories">
 +
                                     
 +
                                          <a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#intro">Introduction</a>
 +
                                          <a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#cth">CHALMERS</a></li>
 +
                                          <a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#svs">Supervisors</a></li>
 +
                                          <a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us#sts">Students</a>
 +
       
 +
  </div>
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                                <div class="list_border"></div> 
 +
                       
 +
                              <div class="Title1">FUSS</div>
 +
                           
 +
                                <div class="left_grad">
 +
                                  <div class="categories">
 +
                                   
 +
                                            <a href="#">What's all about?</a>
 +
                                            <a href="#">Background</a>
 +
                                            <a href="#">Protein Fusion</a>
 +
                                            <a href="#">Methods</a>
 +
                                            <a href="#">Preliminary Results</a>
 +
                                       
 +
                                  </div>
 +
                               
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                                </div>
 +
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 +
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+
<div id="Title2"><ul id="sddm">
-
 
+
    <li><a href="https://2010.igem.org/Team:Gothenburg-Sweden">Home</a></li>
-
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+
    <li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project"
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        onmouseout="mclosetime()"  >Project</a>
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 +
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 +
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 +
        <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Project">Description</a>
 +
        <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note">Lab Notes</a>
 +
        <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Results">Results</a>
 +
        </div>
 +
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-
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 +
        <div id="m2"
 +
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 +
<a href="#">Chalmers University of Technology</a>
 +
        <a href="#">Supervisors</a>
 +
        <a href="#">Students</a>
 +
        </div>
 +
    </li>
 +
 
 +
    <li><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Sponsors">Sponsors</a></li>
 +
    <li id="active"><a href="https://2010.igem.org/Team:Gothenburg-Sweden/Contact">Contact Us</a></li>
 +
</ul>
 +
<div style="clear:both"></div></div>
 +
<div id="text">
 +
</br>
 +
 +
                        <div class="logo"> <img src="https://static.igem.org/mediawiki/2010/a/a5/Chalmers_c_logo.jpg" width="133" height="152" align="left"></div>
 +
                   
 +
                              <p>
 +
                                <strong>CHALMERS UNIVERSITY OF TECHNOLOGY, GOTHENBURG</strong></p>
 +
                       
 +
                              <p>We are a team of 8 students from Chalmers University of Technology    who will represent Gothenburg, SWEDEN in this year’s iGEM competition.     We have started with a promising idea that combines cutting edge    technologies available in the field of Synthetic Biology. Our research    basically aims to constructing an optical reporter mechanism for    cellular stress by tagging the stress activated SNF1 complex in yeast    with fluorescent markers.<br>
 +
                                  <br>
 +
                              The project is performed with two main experimental  pathways: Both  experimental setups will utilize FRET to visualize the  conformational  change that is the result SNF1 activation. The first  approach consists  of fusing fluorescent two proteins, EYFP and ECFP,  with the SNF1  complex. The principal is that when the protein is  activated it  undergoes a conformational change and this should be  indicated by a  change in the FRET-signal. The second approach utilizes  a SAMS-peptide  with fluorescent proteins fused to each end. The  SAMS-peptide will be  phosphorylated by the active SNF1-complex and  undergo a conformational  change that again will be indicated by the  FRET-signal.<br>
 +
  <br>
 +
                                The long term ambition of this project it is to use the  results in  the pharmaceutical industry when performing high-throughput  screening  for new substances or finding the correct drug  concentrations to use.  The yeast cells with the modified SNF1-complex  can be moved through a  micro-fluidic system, gradually exposing them  to an array of substances  or a concentration gradient thereby easily  finding at which  concentration or by what substance the cells are  stressed. <br>
 +
  <br>
 +
                              As of present we have purified the plasmid backbones that  will be  used together with fusion protein insert to transfect the  yeast cells.  The fusion protein primers has arrived and we will start  working with  the fusion PCR as of this week. We have completed the 3D  models of the  fusion proteins and the results look very promising, we  will soon be  able to present docking predictions with the complex.<br>
 +
  <br>
-
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+
<div id="myTitle">Introduction </div>
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+
                            <p><a href="https://2010.igem.org/Team:Gothenburg-Sweden/About_us"><img src="https://static.igem.org/mediawiki/2010/1/1a/Team-gbg.jpg" alt="" align="left" width="174" height="132" style="margin:0 20px 0 0"/> </a><p><span class="style9"><strong>Who we are?</strong> <br/>
-
}
+
                            Left to right: Per Sunnerhagen, Markus Wilhelmsson, Malin Lindén, Karl Almén Burman, Adnan Kadic, Katarina Risö, Peidi Liu, Goutham Vemuri, Lokeshwaran Manoharan, Kemal Sanli, Julia Fransson </span><br /><br />
-
 
+
                            </p>
-
// --------------------------------------------------------------------------------
+
                            <p>&nbsp;</p>
-
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+
                            <p><strong>What we are doing? </strong><br>
-
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+
                            Synthetic readout of cellular stress</p>
-
 
+
                            <p >The cellular stress is sensed  by a key protein called  AMP-activated  protein kinase (AMPK). The AMPK protein  complex is  conserved among all  eukaryotes, including yeast, plants and humans. </p>
-
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+
                 
-
this.a_config = o_parent.a_config[n_order + (this.n_depth ? 3 : 0)];
+
                </div> </div>
-
 
+
-
// return if required parameters are missing
+
-
if (!this.a_config) return;
+
-
 
+
-
// store info from parent item
+
-
this.o_root    = o_parent.o_root;
+
-
this.o_parent  = o_parent;
+
-
this.n_order  = n_order;
+
-
 
+
-
// register in global and parent's collections
+
-
this.n_id = this.o_root.a_index.length;
+
-
this.o_root.a_index[this.n_id] = this;
+
-
o_parent.a_children[n_order] = this;
+
-
 
+
-
// calculate item's coordinates
+
-
var o_root = this.o_root,
+
-
a_tpl  = this.o_root.a_tpl;
+
-
 
+
-
// assign methods
+
-
this.getprop  = mitem_getprop;
+
-
this.getstyle = mitem_getstyle;
+
-
this.upstatus = mitem_upstatus;
+
-
 
+
-
this.n_x = n_order
+
-
? o_parent.a_children[n_order - 1].n_x + this.getprop('left')
+
-
: o_parent.n_x + this.getprop('block_left');
+
-
 
+
-
this.n_y = n_order
+
-
? o_parent.a_children[n_order - 1].n_y + this.getprop('top')
+
-
: o_parent.n_y + this.getprop('block_top');
+
-
 
+
-
// generate item's HMTL
+
-
document.write (
+
-
'<div id="e', o_root.n_id, '_', this.n_id, 'o" class="', this.getstyle(0, 0), '"',
+
-
(this.a_config[2] && this.a_config[2]['tt'] ? ' title="' + this.a_config[2]['tt'] + '"' : ''),
+
-
' style="position:absolute;top: ', this.n_y, 'px;left:', this.n_x, 'px;width:',
+
-
this.getprop('width'), 'px;height:', this.getprop('height'), 'px;visibility:hidden;',
+
-
' z-index: ', this.n_depth, ';cursor:', (this.a_config[1] ? 'pointer' : 'default'), '" ',
+
-
'onclick="return A_MENUS[', o_root.n_id, '].onclick(', this.n_id,
+
-
');" onmouseout="A_MENUS[', o_root.n_id, '].onmouseout(', this.n_id,
+
-
');" onmouseover="A_MENUS[', o_root.n_id, '].onmouseover(',
+
-
this.n_id, ');" onmousedown="A_MENUS[', o_root.n_id, '].onmousedown(',
+
-
this.n_id, ');"><div id="e', o_root.n_id, '_',
+
-
this.n_id, 'i" class="' ,this.getstyle(1, 0), '">',
+
-
this.a_config[0], "</div></div>"
+
-
);
+
-
 
+
-
this.e_ielement = document.getElementById('e' + o_root.n_id + '_' + this.n_id + 'i');
+
-
this.e_oelement = document.getElementById('e' + o_root.n_id + '_' + this.n_id + 'o');
+
-
 
+
-
this.b_visible = !this.n_depth;
+
-
 
+
-
// no more initialization if leaf
+
-
if (this.a_config.length < 4)
+
-
return;
+
-
 
+
-
// node specific methods and properties
+
-
this.a_children = [];
+
-
 
+
-
// init downline recursively
+
-
for (var n_order = 0; n_order < this.a_config.length - 3; n_order++)
+
-
new menu_item(this, n_order);
+
-
}
+
-
 
+
-
// --------------------------------------------------------------------------------
+
-
// reads property from template file, inherits from parent level if not found
+
-
// ------------------------------------------------------------------------------------------
+
-
function mitem_getprop (s_key) {
+
-
 
+
-
// check if value is defined for current level
+
-
var s_value = null,
+
-
a_level = this.o_root.a_tpl[this.n_depth];
+
-
 
+
-
// return value if explicitly defined
+
-
if (a_level)
+
-
s_value = a_level[s_key];
+
-
 
+
-
// request recursively from parent levels if not defined
+
-
return (s_value == null ? this.o_parent.getprop(s_key) : s_value);
+
-
}
+
-
// --------------------------------------------------------------------------------
+
-
// reads property from template file, inherits from parent level if not found
+
-
// ------------------------------------------------------------------------------------------
+
-
function mitem_getstyle (n_pos, n_state) {
+
-
 
+
-
var a_css = this.getprop('css');
+
-
var a_oclass = a_css[n_pos ? 'inner' : 'outer'];
+
-
 
+
-
// same class for all states
+
-
if (typeof(a_oclass) == 'string')
+
-
return a_oclass;
+
-
 
+
-
// inherit class from previous state if not explicitly defined
+
-
for (var n_currst = n_state; n_currst >= 0; n_currst--)
+
-
if (a_oclass[n_currst])
+
-
return a_oclass[n_currst];
+
-
}
+
-
 
+
-
// ------------------------------------------------------------------------------------------
+
-
// updates status bar message of the browser
+
-
// ------------------------------------------------------------------------------------------
+
-
function mitem_upstatus (b_clear) {
+
-
window.setTimeout("window.status=unescape('" + (b_clear
+
-
? ''
+
-
: (this.a_config[2] && this.a_config[2]['sb']
+
-
? escape(this.a_config[2]['sb'])
+
-
: escape(this.a_config[0]) + (this.a_config[1]
+
-
? ' ('+ escape(this.a_config[1]) + ')'
+
-
: ''))) + "')", 10);
+
-
}
+
-
 
+
-
// --------------------------------------------------------------------------------
+
-
// that's all folks
+
-
var MENU_ITEMS =
+
-
[['Home','https://2010.igem.org/Team:Gothenburg-Sweden'],
+
-
['Project', 'https://2010.igem.org/Team:Gothenburg-Sweden/Project', null,
+
-
    ['Description','https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note', null,
+
-
['Aim','https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#aim', null],
+
-
['Theoretic Background','https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#bkg', null],
+
-
['Methods','https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#method', null],
+
-
['Techniques','https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#tech', null],
+
-
['SNF1 & AMPK','https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#snf1', null],
+
-
['Biobricks','https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#bricks', null],
+
-
['Possible Applications','https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#app', null]],
+
-
['Lab Notes','https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note'],
+
-
['Results','https://2010.igem.org/Team:Gothenburg-Sweden/Results']],  
+
-
['About Us','https://2010.igem.org/Team:Gothenburg-Sweden/About_us', null,
+
-
    ['Supervisors', 'https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#aim',null],
+
-
['Students', 'https://2010.igem.org/Team:Gothenburg-Sweden/Project/more#aim', null] ],
+
-
    ['Sponsors','https://2010.igem.org/Team:Gothenburg-Sweden/Sponsors'],
+
-
['Contact Us','https://2010.igem.org/Team:Gothenburg-Sweden/Contact']];
+
-
 
-
// level scope settins structure
 
-
var MENU_TPL = [
 
-
// root level configuration (level 0)
 
-
{
 
-
// item sizes
 
-
'height': 24,
 
-
'width': 155,
 
-
// absolute position of the menu on the page (in pixels)
 
-
// with centered content use Tigra Menu PRO or Tigra Menu GOLD
 
-
'block_top':  315,
 
-
'block_left': 279,
 
-
// offsets between items of the same level (in pixels)
 
-
'top':  0,
 
-
'left': 131,
 
-
// time delay before menu is hidden after cursor left the menu (in milliseconds)
 
-
'hide_delay': 200,
 
-
// submenu expand delay after the rollover of the parent
 
-
'expd_delay': 200,
 
-
// names of the CSS classes for the menu elements in different states
 
-
// tag: [normal, hover, mousedown]
 
-
'css' : {
 
-
'outer' : ['m0oout', 'm0oover'],
 
-
'inner' : ['m0iout ', 'm0iover']
 
-
}
 
-
},
 
-
// sub-menus configuration (level 1)
 
-
// any omitted parameters are inherited from parent level
 
-
{
 
-
'height': 24,
 
-
'width': 170,
 
-
// position of the submenu relative to top left corner of the parent item
 
-
 
-
'block_top': 25,
 
-
'block_left': 0,
 
-
'top': 23,
 
-
'left': 0,
 
-
'css' : {
 
-
'outer' : ['m1oout', 'm1oover'],
 
-
'inner' : ['m1iout', 'm1iover']
 
-
}
 
-
},
 
-
// sub-sub-menus configuration (level 2)
 
-
{
 
-
'block_top': 5,
 
-
'block_left': 160
 
-
}
 
-
// the depth of the menu is not limited
 
-
// make sure there is no comma after the last element
 
-
];
 
-
 
-
 
-
new menu (MENU_ITEMS, MENU_TPL);
 
-
 
-
// If you don't see the menu then check JavaScript console of the browser for the error messages
 
-
// "Variable is not defined" error indicates that there's syntax error in that variable's definition
 
-
// or the file with that variable isn't properly linked to the HTML document
 
-
//-->
 
-
    </script>
 
-
        <div id="mainbg">
 
-
         
 
-
            <div id="header" align="left">
 
-
<img src="https://static.igem.org/mediawiki/2010/4/44/HeaderGo.jpg">
 
-
</div>
+
-
                 </div>
+
<div id="right">
-
 
+
                 <div class="Title3">Lab Notes</div>
 +
                         
 +
                             
 +
                                      <div id="lab">
 +
                                        <div class="numleft">
 +
                                          <p>11</p>
 +
                                        </div>
 +
                                          <div class="newsright"><a href="#">August 11, 2010</a>
 +
                                          <p> The PCR reaction from yesterday was checked on a gel. α should be ... </p>
 +
                                          </div>
 +
 
 +
                                          <div class="read"><a href="#">read more</a></div>
 +
                                          <div style="clear: both"></div>
 +
                                   
 +
           
 +
                               
 +
    </div>   
 +
  <div class="Title3">News</div>
 +
                              <div id="news">
 +
                                    <p>Göteborgs-Posten, a major daily newspaper in Sweden with the second largest national circulation, published an article involving the interview with our team...
 +
                                    <br><br> </p>
 +
<div class="read"><a href="#">read more</a></div>
 +
                                          <div style="clear: both"></div>
 +
                                </div>
 +
                     
 +
 
 +
 +
</div>
   
   
-
                <!-- header ends -->
+
  <div id="myfooter"> <p>   <a href="#">Home</a> | <a href="#">CTH</a> | <a href="#">Sponsors</a> | <a href="#">About Us</a> | <a href="#">Contact Us</a></p> <p>Chalmers University of Technology, Gothenburg, SWEDEN</p>
-
                <!-- content begins -->
+
-
                <div id="main">
+
-
                    <div id="content">
+
-
                        <div id="right">
+
-
                            <h2>FUSS</h2>
+
-
                            <div class="left_bg">
+
-
                                <div class="left_grad">
+
-
                                    <div class="categories">
+
-
                                        <ul>
+
-
                                            <li><a href="#">What's all about?</a></li>
+
-
                                            <li><a href="#">Theoretical Background</a></li>
+
-
                                            <li><a href="#">Fusion Protein Design</a></li>
+
-
                                            <li><a href="#">Experimental Methods</a></li>
+
-
                                            <li><a href="#">Preliminary Results</a></li>
+
-
                                        </ul>
+
-
                                    </div>
+
-
                                </div>
+
-
                            </div>
+
-
                            <div class="list_border"></div>
+
-
                            <h2>Team</h2>
+
              </div>
-
                            <div class="left_bg">
+
</div></div>  
-
                                <div class="left_grad">
+
</body>
-
                                  <div class="categories"> 
+
-
    <ul>
+
-
                                            <li><a href="#">Introduction</a> </li>
+
-
<li><a href="#">Chalmers University of Technology</a></li>
+
-
                                            <li><a href="#">Supervisors</a></li>
+
-
                                            <li><a href="#">Students</a></li>
+
-
                                         
+
-
                                         
+
-
                                        </ul>
+
-
 
+
-
                                  </div>
+
-
</div>
+
-
                              </div>  
+
-
                                  <div class="list_border"></div> 
+
-
                                </div>
+
-
                         
+
-
                           
+
-
                        </div>
+
-
 
+
-
                        <div id="left">
+
-
                            <h2>News</h2>
+
-
                            <div class="left_bg">
+
-
                                <div class="left_grad">
+
-
                                    <div class="categories">
+
-
                                        <ul>
+
-
                                            <li><a href="#">Göteborgs-Posten to publish...</a></li>
+
-
                                            <li><a href="#">Team members reunite...</a></li>
+
-
                                            <li><a href="#">Sponsor deal set...</a></li>
+
-
                                            <li><a href="#">Yeast resistance for sporulation... </a></li>
+
-
                                       
+
-
                                        </ul>
+
-
                                    </div>
+
-
                                </div>
+
-
                          </div>
+
-
                       
+
-
                            <div class="list_border"></div>
+
-
 
+
-
                            <h2>Lab Notes</h2>
+
-
                            <div class="left_bg">
+
-
                              <div class="left_grad">
+
-
                                    <div>
+
-
                                        <div class="numleft">
+
-
                                          <p>11</p>
+
-
                                        </div>
+
-
                                        <div class="newsright"><a href="#">August 11, 2010</a>
+
-
                                            <p>The PCR reaction from yesterday was checked on a gel. α should be ... </p>
+
-
                                        </div>
+
-
                                        <div class="read"><a href="#">read more</a></div>
+
-
                                        <div style="clear: both"></div>
+
-
                                    </div>
+
-
                                    <div>
+
-
                                        <div class="numleft"><p>10</p></div>
+
-
                                        <div class="newsright"><a href="#">August 10, 2010</a>
+
-
                                            <p>Checked the transformation plates but nothing had grown. Prepared PCR reaction Γ ...  </p>
+
-
                                        </div>
+
-
                                     
+
-
                                   
+
-
                               
+
-
                                    </div>  <div class="read"><a href="#">read more</a></div>
+
-
<div class="list_border"></div>
+
-
 
+
-
                              </div>
+
-
                            </div>
+
-
                        </div>
+
-
+
-
                        <div id="center">
+
-
                            <h1>Preliminary</h1>
+
-
                            <div class="text">
+
-
                             
+
-
                              <strong><p>Fluorescent  Proteins:</p></strong>
+
-
    <p>Green Fluorescent Protein <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#FP">(read more)</a></p>
+
-
<strong><p>Plasmid backbone:</p></strong>
+
-
                    <p>pSP-GM1 <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#plasmid">(read more)</a></p>
+
-
    <strong><p>SNF1  (modified subunits):</p></strong>
+
-
                    <p>Snf1 (alfa), Snf4 (gamma) <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#SNF1">(read more)</a></p>
+
-
    <strong><p>FP  positions:</p></strong>
+
-
                    <p>N-terminal alfa with N-terminal gamma, N-terminal gamma with C-terminal gamma <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#position">(read more)</a></p>
+
-
    <strong><p>Primer  design:</p></strong>
+
-
                    <p>Fusion primers with restiriction enzyme sites included at very ends, each annealing part was desinged with an melting temperature of around 60 C <a href="https://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#primer">(read more)</a></p>
+
-
 
+
-
                          </div>
+
-
                         
+
-
                            <h1>Lab work by date </h1>
+
-
                            <div class="text">    <ul><li><p> <strong>2010-08-11</strong><br>
+
-
                              Malin and Katarina<br>
+
-
                              <br>
+
-
                              The PCR reaction from yesterday was checked on a gel. α should be around 1500 bp but only a band around 750 bp was seen. We had a long meeting with supervisor Per about PCR reactions and the next couple of weeks in the lab. <br>
+
-
                              <br>
+
-
                              Karl and Kemal <br>
+
-
                              <br>
+
-
                              Ran PCR reaction α with different settings. <br>
+
-
                              <strong>α1: </strong>regular settings<br>
+
-
                              <strong>α2: </strong>10 x diluted template 2<br>
+
-
                              <strong>α3: </strong>increased annealing time from 30 s to 60 s<br>
+
-
                              <strong>α4: </strong>decreased annealing temperature<br>
+
-
                              <br>
+
-
                              After PCR, the products were run on a gel. α1, α3 and α 4 had a band around 750 bp but nothing on α2. </p></li><br>
+
-
                              <br>
+
-
                              <li><p> <strong>2010-08-10</strong><br>
+
-
                              Peidi and Lokesh<br>
+
-
                              <br>
+
-
                              Checked the transformation plates but nothing had grown. Prepared PCR reaction Γ. <br>
+
-
                              <br>
+
-
                              Katarina and Kemal<br>
+
-
                              <br>
+
-
                              PCR product Γ was verified on a 0.7 % agarose gel. There was a band around 750 bp as expected and it was purified using the regular protocol. A new phusion mastermix was made and PCR reaction α was run with Γ as one of the template. Regular phusion PCR settings was used. <br>
+
-
                              <br></p></li>
+
-
                              <li><p><strong>2010-08-09</strong><br>
+
-
                              Malin and Kemal<br>
+
-
                              <br>
+
-
                              Before checking if the ligation worked we need to transform and amplify in E.coli. Then we can miniprep the ligated plasmid and check on a gel. After that we can digest plasmid with PacI and BcuI and ligate the digested IV. Then another round of transformation and miniprep before we can transform yeast. <br>
+
-
                              <br>
+
-
                              Karl and Lokesh<br>
+
-
                              <br>
+
-
                              Transformed competent E.coli with plasmid ligated with III by heat shock. The cells were incubated at 37 °C over night. <br>
+
-
                              <br></p></li>
+
-
                              <li><p> <strong>2010-08-07</strong><br>
+
-
                              Lokesh<br>
+
-
                              <br>
+
-
                              Moved ligation tube to freezer. <br>
+
-
                              <br></p></li>
+
-
                            <li><p>  <strong>2010-08-06</strong><br>
+
-
                              Malin, Julia and Lokesh<br>
+
-
                              <br>
+
-
                              III and IV were digested 3x protocol and were then put on a 1 % agarose gel together with Γ both for verification and purification. Correct bands were seen for purification except for Γ which was empty. In the verification lanes the digested PCR products were to light to be seen but they were seen in purification lanes. IIId and IVd were purified. <br>
+
-
                              <br>
+
-
                              <strong>IIId: </strong>7.8 ng/µl<br>
+
-
                              <strong>IVd: </strong>11.7ng/µl<br>
+
-
                              <br>
+
-
                              IIId will now be ligated with the cut plasmid (BamHI and HindIII). Ligation performed according to protocol with 156 ng insert and 52 ng plasmid. Ligation was incubated over night in fridge at 4-16° C. <br>
+
-
                              <br>
+
-
                              All 4 sporulation plates were checked but still no tetrads visible. <br>
+
-
                              <br></p></li>
+
-
                              <li><p>  <strong>2010-08-05</strong><br>
+
-
                              Malin and Julia<br>
+
-
                              <br>
+
-
                              III and IV were digested using the regular protocol. <br>
+
-
                              <strong>III: </strong>HindIII and BamHI works with Buffer B<br>
+
-
                              <strong>IV: </strong>PacI and BcuI works with Buffer M<br>
+
-
                              <br>
+
-
                              A 1% gel was run with uncut and digested III and IV, control, I and Β. I worked well and will be purified. The Β lane showed no bands. None of the digested products were visible on the gel so a new gel was run trying to verify this result. The control shows a band around 1700 which is weird. This band is also visible on the III uncut but together with a band around 750 bp. No bands at the IV which is very strange since this PCR product has been verified before. Have something gone wrong with the purification? <br>
+
-
                              <br>
+
-
                              As a result PCR reactions III and IV will be redone. New PCR reactions of Γ and α were also performed in the same block. <br>
+
-
                              <br>
+
-
                              Karl and Kemal<br>
+
-
                              <br>
+
-
                              Reactions of Β were made. Β1 is a continuation of the 100 cycle PCR this time with primers. Β2 uses a megaprimer approach. <br>
+
-
                              <br>
+
-
                              All the PCR products were run on a 1 % agarose gel. III, IV and Γ were alright. α and Β2 showed a band around 750 bp. <br>
+
-
                              <strong>III: </strong>531.3 ng/µl<br>
+
-
                              <strong>IV: </strong>263.9 ng/µl<br>
+
-
                              <br></p></li>
+
-
                              <li><p> <strong>2010-08-04</strong><br>
+
-
                              Julia and Karl<br>
+
-
                              <br>
+
-
                              PCR products Β were loaded on a 0.7 % gel. No correct bands, only amplified templates around 750 bp and something else around 500 bp. <br>
+
-
                              <br>
+
-
                              PCR of SAMS will also be redone from scratch. PCR reactions 1, 2 and C are done, then loaded on a gel (2 % gel and low range ladder) and purified. <br>
+
-
                              <strong>1: </strong>8.2 ng/µl<br>
+
-
                              <strong>2: </strong>7.8 ng/µl<br>
+
-
                              <strong>C: </strong>4.9 ng/µl<br>
+
-
                              Samples diluted to 1 ng/µl. <br>
+
-
                              <br>
+
-
                              PCR of Β was done again but this time in two steps and with the Finnzymes Tm which corresponds to an annealing temp at 85 °C. Firstly 10 cycles without primers were performed. <br>
+
-
                              <br>
+
-
                              Kemal and Lokesh<br>
+
-
                              <br>
+
-
                              Second round of PCR with primers were performed. Then new PCR reactions amplifying III and IV were done and also reaction I. <br>
+
-
                              <strong>III: </strong>572 ng/µl<br>
+
-
                              <strong>IV: </strong>660 ng/µl<br>
+
-
                              <br>
+
-
                              The sporplates were checked and very few tetrads were seen. <br>
+
-
                              <br>
+
-
                              Β samples were put on a gel but no correct bands visible. We suspect that the reverse primer is attaching in the wrong place since YFP and CFP is so similar which then would lead to 750 bp fragment. <br>
+
-
                              <br></p></li>
+
-
                              <li><p>  <strong>2010-08-03</strong><br>
+
-
                              Malin and Lokesh<br>
+
-
                              <br>
+
-
                              PCR products II, III and IV were verified and purified from a 1 % agarose gel. III did not work and was not purified. <br>
+
-
                              <br>
+
-
                              Concentrations: <br>
+
-
                              <strong>II: </strong>6.2 ng/µl <br>
+
-
                              <strong>IV: </strong>5.6 ng/µl<br>
+
-
                              II and IV were diluted to 1 ng/µl. <br>
+
-
                              <br>
+
-
                              The new sporplates were examined but no tetrads visible. To check further the cells were stained with DAPI and examined in fluorescence microscope. Still no success. <br>
+
-
                              <br>
+
-
                              New PCR reactions were prepared. III was redone and Β was also done. Same settings as yesterday. <br>
+
-
                              <br>
+
-
                              Kemal and Karl<br>
+
-
                              <br>
+
-
                              PCR products were loaded on a 1% agarose gel for verification and purification. III worked this time and was purified from the gel using the protocol from QIAquick Gel Extraction Kit. <br>
+
-
                              <strong>III: </strong>6.4 ng/µl<br>
+
-
                              <br>
+
-
                              New PCR of Β was run with two different block settings. <br>
+
-
                              <strong>Block A: </strong>increased annealing time to 1 min. <br>
+
-
                              <strong>Block B: </strong>increased annealing time to 58 °C. <br>
+
-
                              <br></p></li>
+
-
                              <li><p> <strong>2010-08-02</strong><br>
+
-
                              Kemal, Lokesh and Karl<br>
+
-
                              <br>
+
-
                              PCR products A, B and 3 were loaded on a gel and then purified from the gel using the protocol from QIAquick Gel Extraction Kit. α and IV were also run on a gel but only showed strong bands corresponding to amplified templates. On the α lane however a very light band was seen at the correct length. This will be further investigated. <br>
+
-
                              <br>
+
-
                              Malin and Julia<br>
+
-
                              <br>
+
-
                              The purified PCR products were measured with nanodrop: <br>
+
-
                              <strong>A1: </strong>11.8 ng/µl<br>
+
-
                              <strong>A2: </strong>10.2 ng/µl<br>
+
-
                              <strong>B1: </strong>9.6 ng/µl<br>
+
-
                              <strong>B2: </strong>17.1 ng/µl<br>
+
-
                              <strong>31: </strong>15.0 ng/µl<br>
+
-
                              <strong>32: </strong>16.3 ng/µl<br>
+
-
                              <br>
+
-
                              A, B and 3 were diluted to 1 ng/µl. <br>
+
-
                              <br>
+
-
                              PCR products 4 and 5 were loaded on a 1 % agarose gel together with IV that should be controlled. PCR products 4 and 5 were correct so they were purified from the gel using the protocol from QIAquick Gel Extraction Kit. IV however was not correct. Only a band around 750 bp was visible. <br>
+
-
                              <br>
+
-
                              Concentrations: <br>
+
-
                              <strong>4: </strong>10.8 ng/µl<br>
+
-
                              <strong>5: </strong>10.4 ng/µl<br>
+
-
                              4-5 were diluted to 1 ng/µl. <br>
+
-
                              New PCR reactions were started (II, III and IV). PCR settings that works well with the Phusion enzyme was set: <br>
+
-
                              Initial denaturation: 98 °C 3 min<br>
+
-
                              Denaturation: 98 °C 10 s<br>
+
-
                              Annealing: 55 °C 30 s<br>
+
-
                              Elongation: 72 °C 1.5 min+ 1s/cycle<br>
+
-
                              30 cycles<br>
+
-
                              Final extension: 72 °C 10 min<br>
+
-
                              4°C forever <br>
+
-
                              <br>
+
-
                              <strong>2010-07-30</strong><br>
+
-
                              Julia and Katarina<br>
+
-
                              <br>
+
-
                              The concentration of the purified plasmids were measured with nanodrop: <br>
+
-
                              <strong>P1: </strong>1.7 ng/µl<br>
+
-
                              <strong>P2: </strong>3.5 ng/µl<br>
+
-
                              <strong>P3: </strong>25.9 ng/µl<br>
+
-
                              <strong>P4: </strong>42.8 ng/µl<br>
+
-
                              This is clear evidence that water should be used for eluation in the future! <br>
+
-
                              <br>
+
-
                              To make sure that we have the right products after purification a 1 % agarose gel was run with fastruler DNA ladder. The gel verified the plasmid size though the bands were light. <br>
+
-
                              <br>
+
-
                              PCR product III was evaporated since it was so dilute. New concentration: <br>
+
-
                              <strong>IIId: </strong>35 ng/µL<br>
+
-
                              <br>
+
-
                              The digested PCR product III and the digested plasmid were ligated with T4 ligase. The DNA concentration in total should not exceed 1 µg. The ratio insert:vector should be 3:1. <br>
+
-
                              <br>
+
-
                              <strong>Ligation protocol: </strong><br>
+
-
                              150 ng insert<br>
+
-
                              50 ng vector<br>
+
-
                              3 µl 10x T4 buffer<br>
+
-
                              2.5 µl (2.5 U, between 1-5 U) T4 ligase<br>
+
-
                              Adjust volume with water up to 30 µl. Mix and incubate in 4-16 °C (refrigerator) overnight. <br>
+
-
                              The newly plated sporplates were moved to 30°C. The old sporplates were again studied in the microscope and no tetrads were seen. <br>
+
-
                              <br>
+
-
                              Kemal and Lokesh<br>
+
-
                              <br>
+
-
                              Since we have had a lot of trouble with the longer fusions we will start over from scratch with a new high fidelity polymerase called “Finnzumes Phusion”. New PCR reactions were run (A, B, 3, 4 and 5). IV and α were also performed using alternative settings (2 different PCR reactions described before) but with “old” templates. <br>
+
-
                              <br></p></li>
+
-
                              <li><p><strong>2010-07-29</strong><br>
+
-
                              Julia and Katarina<br>
+
-
                              <br>
+
-
                              PCR product III is the final fusion protein where Snf4 is tagged with yfp. To be able to insert the construct into the plasmid we must digest it. <br>
+
-
                              <br>
+
-
                              Digestion: Mix DNA, buffer and water. Last add enzyme and mix. Incubate at 37° C for 1 hour. <br>
+
-
                              1 µg DNA<br>
+
-
                              2.5 µl Buffer<br>
+
-
                              1 U Enzyme<br>
+
-
                              Up to 25 µl H2O<br>
+
-
                              For PCR product III, the restriction enzymes BamHI and HindIII are used together with buffer B. After digestion (the protocol was scaled 3x) the sample was loaded on a 1 % agarose gel in 6 lanes with 1 kb ladder. The bands were cut out and weighed. The samples were purified from the gel using the protocol from QIAquick Gel Extraction Kit. The DNA was eluated with water. <br>
+
-
                              <br>
+
-
                              Concentration: <br>
+
-
                              <strong>IIId: </strong>5 ng/µl (Total product 160 µl= 800 ng) <br>
+
-
                              <br>
+
-
                              Kemal and Lokesh<br>
+
-
                              <br>
+
-
                              The plasmids were also digested with BamHI and HindIII according to the same protocol as above. P1 and P2 were eluated with buffer EB and P3 and P4 with water. <br>
+
-
                              <br>
+
-
                              2 new sporulation plates were prepared by plating the diploid yeast cells on spore plates and kept at room temperature. <br>
+
-
                              <br></p></li>
+
-
                                <li><p><strong>2010-07-28</strong><br>
+
-
                              Julia and Katarina<br>
+
-
                              <br>
+
-
                              The PCR products were run on a 1 % gel with 1 kb ladder. Γ may have worked. α and Β had a band around 750 bp and a weird light band around 500 bp. They obviously did not work. IV did not work at all. <br>
+
-
                              <br>
+
-
                              The sporulation plates were checked for spores. Cells were studied under a microscope. No tetrads visible. <br>
+
-
                              <br></p></li>
+
-
                              <li><p> <strong>2010-07-27</strong><br>
+
-
                              Julia and Katarina<br>
+
-
                              <br>
+
-
                              A 1.5 % gel was run with all of the fragments around 700 bp to control the validity of the PCR reactions. A low range ladder was used. PCR products 1-5 seems good. The results from yesterday were confirmed. <br>
+
-
                              <br>
+
-
                              A new PCR design was made with α, Β, Γ and IV. PCR was done in two steps to increase the chance of success for longer fusions. <br>
+
-
                              1. 7 cycles with short annealing time. <br>
+
-
                              2. 20 cycles with long annealing time (all other settings kept standard). <br>
+
-
                              <br></p></li>
+
-
                                <li><p><strong>2010-07-26</strong><br>
+
-
                              <br>
+
-
                              A new experiment were designed called Γ which is the same as I but with longer annealing region. Also an experiment called Δ were designed which corresponds to II. <br>
+
-
                              <br>
+
-
                              Julia and Katarina<br>
+
-
                              <br>
+
-
                              PCR reactions were run with samples of 4, IV and Γ. IV was tried with two different PCR settings: one regular and one with two runs of PCR, the first without primers and only 5 cycles and the second one with regular settings. <br>
+
-
                              <br>
+
-
                              Kemal and Lokesh<br>
+
-
                              <br>
+
-
                              The PCR products were controlled on a 0.7% agarose gel. Only 4 worked. Γ was empty and IV had bands in the wrong place (amplified template). <br>
+
-
                              <strong>4: </strong>534 ng/ µl </p></li>
+
-
                                <li><p>  <strong>2010-07-22</strong><br>
+
-
                                      Karl, Lokesh and Kemal
+
-
                                      </br></br>
+
-
                                      A new mastermix was prepared using a different enzyme. Then the  experiments from yesterday was redone using regular PCR settings: <br>
+
-
                                      A, 5, 2, I<br>
+
-
                                      Then some more experiments of IV was redone: <br>
+
-
                                      2a, 2b and 2c<br>
+
-
                                      The new primer 14 had arrived so PCR reaction 4 was also redone. <br>
+
-
                                      <br>
+
-
                                      All the PCR products were run on a 0.7 % agarose gel with GeneRuler 1 kb  DNA ladder. The results were inconclusive. None of the expected bands  were received.  At this point we are not sure what to do since none of  the PCR reactions worked. <br>
+
-
                                      <br>
+
-
                                      </p>
+
-
                                </li>
+
-
                                <li> <p> <strong>2010-07-21</strong><br>
+
-
                                  Malin, Julia, Karl and Lokesh<br>
+
-
                                  <br>
+
-
                                  <strong>Verification of the PCR products </strong>α1, α2, α3, α4, α5 and αK by running on a thin 0.7 % agarose gel (8 µl sample+ 2 µl loading buffer, GeneRuler 1 kb DNA ladder). <br>
+
-
                                  <br>
+
-
                                  The gel didn’t show the expected band lengths. A band around 700 bp  could be seen, most strongly for α4 and α5.  We suspect that there is  something wrong with the ladder because we get inconclusive results.  Since none of the PCR reactions for the last couple of days have worked  we will redo the templates of α and IV: I, 2, 5 and A. These PCR  reactions have worked before. <br>
+
-
                                  <br>
+
-
                                  <strong>Expected fragment length: </strong><br>
+
-
                                  A.2: 1938 bp<br>
+
-
                                  5.2: 757 bp<br>
+
-
                                  2.2: 762 bp<br>
+
-
                                  I.2: 768 bp<br>
+
-
                                  <br>
+
-
                                  The PCR products were run on a thin 0.7 % agarose gel. We used both the  FastRuler High Range Ladder and the GeneRuler 1 kb DNA ladder. 5.2 and  2.2 showed the expected bands around 750 bp. I.2 and A.2 did not work at  all, nothing visible on the gel.  We also run some of the old PCR  products to see what the result would be using a new ladder. We run α2,  α5, 4c (IV) and 4c*(IV). Here some template amplifications were visible  but sadly none of the expected fragment lengths. Both ladders showed the  same result so there is probably nothing wrong with the GeneRuler  ladder except that it is not showing strong color. <br></p></li>
+
-
                                  <li><p><strong>2010-07-20</strong><br>
+
-
                                  Malin, Julia, Lokesh and Karl<br>
+
-
                                  <br>
+
-
                                  <strong>Verification of yesterdays PCR reactions </strong>by running on a 0.7 % agarose gel (8 µl sample+ 2 µl loading buffer, GeneRuler 1 kb DNA ladder) <br>
+
-
                                  <br>
+
-
                                  None of the reactions worked. <br>
+
-
                                  <br>
+
-
                                  Gel 1 (Block A): Nothing visible on gel, ladders only very light so  these samples were run on a new 0.7 % agarose gel but this time thinner  to get better resulotion (0.58 g agarose + 80 ml TBE). This time no  expected fragments were seen, but bands corresponding to primer dimers  were clearly seen. <br>
+
-
                                  Gel 2 (Block B): A few small bands were seen but no bands of expected length. <br>
+
-
                                  <br>
+
-
                                  We decided to stop working on IV until proper feedback on the experimental setup was received. Instead we continued with α.<br>
+
-
                                  <br>
+
-
                                  <strong>Doing another PCR round of α (SAMS peptide): </strong> <br>
+
-
                                  <strong>α: PCR fusion of cfp-SAMS and yfp: </strong>Adds BamHI on N-terminus of cfp and HindIII on C-terminus of yfp and keeps the stop codons. Expected fragment length: 1502 bp. <br>
+
-
                                  <br>
+
-
                                  α1: regular protocol. <br>
+
-
                                  α2: regular protocol. <br>
+
-
                                  α3: 1.25 µl extra primer 9.<br>
+
-
                                  α4: 1.25 µl extra primer 12. <br>
+
-
                                  α5: 1.25 µl extra primer 9 and 1.25 µl primer 12. <br>
+
-
                                  αK: Control<br>
+
-
                                  Regular PCR-settings</p></li>
+
-
                                <li><p> <strong>2010-07-19</strong><br>
+
-
                                  Karl, Peidi and Kemal<br>
+
-
                                  <br>
+
-
                                  <strong>Doing a new experimental setup for IV: </strong><br>
+
-
                                  <strong>IV. PCR fusion of cfp and Snf1: </strong>Adds SpeI on N-terminus of cfp and PacI on C-terminus of Snf1 and keeps the stop codon. Expected fragment length: 2643 bp. <br>
+
-
                                  <br>
+
-
                                  13 different tubes were prepared and run at two different PCR-settings.  Block A: Regular settings except double annealing time (1 min). Block B:    Regular settings except 58° C annealing temperature. <br>
+
-
                                  <br>
+
-
                                  C : Control<br>
+
-
                                  1a: same as 2* (template 5 5 x diluted) <br>
+
-
                                  1b. same as 3* (template 5 10 x diluted) <br>
+
-
                                  1c: same as 4* (template 5 100x diluted) <br>
+
-
                                  2a: increased product A concentration to 5 ng/µl. <br>
+
-
                                  2b: increased product A concentration to 10 ng/µl. <br>
+
-
                                  3a: same as 5* (2x primer 7) <br>
+
-
                                  3b: same as 6* (template 5 diluted 5x, 2x primer 7) <br>
+
-
                                  3c: same as 7* (template 5 diluted 10x, 2x primer 7) <br>
+
-
                                  3d same as 8* (template 5 diluted 100x, 2x primer 7) <br>
+
-
                                  4a: 2x primer 7. <br>
+
-
                                  4b: 2x primer 7, 5x template A. <br>
+
-
                                  4c: 2x primer 7, 10 x template A. <br></p></li>
+
-
                                <li><p> <strong>2010-07-16</strong><br>
+
-
                                  Lokesh, Kemal, Katarina and Peidi<br>
+
-
                                  <br>
+
-
                                  <strong>Verification of PCR </strong>reactions IV* (8 tubes) and α on a 0.7 % agarose gel (5 µl sample+ 2µl loading buffer, GeneRuler 1 kb DNA ladder). <br>
+
-
                                  <br>
+
-
                                  No bands visible. None of the PCR reactions worked, just small bands  visible corresponding to primers. The α PCR reaction messed the tubes  up. That might be an explanation why there were no bands on the gel for  α.  A new experimental setup must be made tomorrow. <br>
+
-
                                  <br>
+
-
                                  PCR products I and 2 were run with a low range ladder to see that those products are ok. That gel was satisfactory. <br>
+
-
                                  <br></p>
+
-
                                  <li><p><strong>2010-07-15</strong><br>
+
-
                                  Malin, Julia, Kemal and Karl<br>
+
-
                                  <br>
+
-
                                  <strong>Concentrations of PCR products: </strong><br>
+
-
                                  4.2. 356.2 ng/µl<br>
+
-
                                  I. 338.3 ng/µl<br>
+
-
                                  II. 303.6 ng/µl<br>
+
-
                                  III. 337.5 ng/µl<br>
+
-
                                  IV. 280.9 ng/µl<br>
+
-
                                  <br>
+
-
                                  All PCR-products were diluted to 1 ng/µl. <br>
+
-
                                  <br>
+
-
                                  <strong>Verified PCR products</strong> I-IV and 4.2 on a 0.7 % Agarose Gel (8 µl sample + 2µl loading buffer, GeneRuler 1 kb DNA ladder) <br>
+
-
                                  PCR products I, II and III were good but 4.2 showed no bands at all.  After primer design control we found a problem with primer 14, which  then was ordered again. IV had only a band around 700 bp instead of  2600.  This might be template 5, which then has been amplified. If the  settings are changed maybe it will work. Firstly the template 5 will be  diluted 0x, 5x, 10x and 100x (4 tubes: 1*, 2*, 3* and 4*) and these  dilutions will also be run with higher primer concentration 7 (4 tubes:  5*, 6*, 7* and 8*).  2.5 µl primer 7 will be added instead of 1.25 µl  primer 7. <br>
+
-
                                  <br>
+
-
                                  New PCR reactions were run: <br>
+
-
                                  <strong>α: PCR fusion of cfp-SAMS and yfp: </strong> Adds BamHI on N-terminus of cfp and HindIII on C-terminus of yfp and keeps the stop codons. Expected fragment length: 1502 bp. <br>
+
-
                                  K4. Control. <br>
+
-
                                  <br>
+
-
                                  <strong>IV *: PCR fusion of cfp and Snf1: </strong>Adds SpeI on N-terminus of cfp and PacI on C-terminus of Snf1 and keeps the stop codon. Expected fragment length: 2643 bp. <br>
+
-
                                  Half volume 25 µl. Regular PCR settings except 56 °C annealing temperature. <br>
+
-
                                  1*;- 8*<br>
+
-
                                  K1*. Control for 1-4*.<br>
+
-
                                  K2*. Control for 5-8*.<br>
+
-
                                  <br></p></li>
+
-
                             
+
-
                                <li> <p><strong>2010-07-14</strong><br>
+
-
                                  Julia and Katarina <br>
+
-
                                  <strong>Ran PCR 1-5.</strong><br>
+
-
                                  1. Amplification of cfp-sams compatible: Add restriction enzyme site BamHI on N-terminus +SAMS on C-terminus.<br>
+
-
                                  Expected fragment length: 748 bp.<br>
+
-
                                  2. Amplification of yfp-SAMS competible: Adds restriction enzyme site for HindIII on C-terminus and SAMS tail on N-terminus. <br>
+
-
                                  Expected fragment length: 762 bp.<br>
+
-
                                  3. Amplification of yfp-snf4 compatible: Adds restriction enzyme site BamHI on N-terminus and Snf4 tail on C-terminus. <br>
+
-
                                  Expected fragment length: 755 bp.<br>
+
-
                                  4. Amplification of cfp-Snf4 compatible: Adds restriction enzyme site HindIII on C-terminus and Snf4 tail on N-terminus. <br>
+
-
                                  Expected fragment length: 769 bp.<br>
+
-
                                  5. Amplification of cfp-snf1 compatible: Adds restriction enzyme site SpeI on N-terminus and Snf1 tail on C-terminus.<br>
+
-
                                  Expected fragment length: 757 bp.<br>
+
-
                                  K2. Control K2.<br>
+
-
                                  <br>
+
-
                                  Julia, Katarina and Malin<br>
+
-
                                  <strong>Verified PCR 1-5:</strong> measured concentration by nanodrop  and checked if the fragments were the correct length by running on a 2 %  agarose gel (8 µl sample+2 µl loading buffer, Fermentas FastRuler Low  Range).<br>
+
-
                                  PCR product 1, 2, 3 and 5 worked and gave expected fragment lengths.  Number 4 however did not work.  Only a fragment around 80 bp was  visible, probably a primer dimer.<br>
+
-
                                  <br>
+
-
                                  <strong>Concentrations:</strong><br>
+
-
                                  1. 443.9 ng/µl<br>
+
-
                                  2. 371.0 ng/µl<br>
+
-
                                  3. 361.7 ng/µl<br>
+
-
                                  4. --<br>
+
-
                                  5. 377.6 ng/µl<br>
+
-
                                  A. 451.1 ng/µl (snf1)<br>
+
-
                                  B. 438.7 ng/µl (snf4)<br>
+
-
                                  C. 408.9 ng/µl (SAMS) <br>
+
-
                                  1-5 and A-C were diluted to 1 ng/µl.<br>
+
-
                                  <br>
+
-
                                  <strong>Ran PCR of I, II, III, IV and 4.</strong><br>
+
-
                                  4.2.  Amplification of cfp-Snf4 compatible: Adds restriction enzyme site  HindIII on C-terminus and Snf4 tail on N-terminus. Expected fragment  length: 769 bp. (second try)<br>
+
-
                                  I. PCR fusion of cfp and SAMS: Adds BamHI on N-terminus of cfp and gives a SAMS C-terminus. Expected fragment length: 768 bp.<br>
+
-
                                  II. PCR fusion of yfp and Snf4 1: Adds BamHI on N-terminus of yfp and removes stop codon. Expected fragment length: 1694 bp.<br>
+
-
                                  III. PCR fusion of yfp and Snf4 2: Adds BamHI on N-terminus of yfp and  adds HindIII on C-terminus of Snf4 plus adds stop codon. Expected  fragment length: 1708 bp. Final Product!<br>
+
-
                                  IV. PCR fusion of cfp and Snf1: Adds SpeI on N-terminus of cfp and PacI  on C-terminus of Snf1 and keeps the stop codon. Expected fragment  length: 2643 bp.<br>
+
-
                                  K3. Control<br>
+
-
                                  <br>
+
-
                                  Karl and Malin<br>
+
-
                                  Transferred diploid cells of snf1Δ mutants to two sporulation plates and incubated in 30° C. (For 2-7 days)<br>
+
-
                                </p></li>
+
-
                             
+
-
                                <li><p><strong>2010-07-13</strong><br>
+
-
                                  Julia and  Lokesh<br>
+
-
                                  Ran the gel  with products of our first three PCRs along with the  templates we made for the  two fluorescent proteins. We also prepared  the sample solutions of DNA  templates with appropriate concentration  (1ng/µl) for the next PCR reactions.<br>
+
-
                                  <strong>Verified</strong> the PCR products and the digested miniprepped    EYFP and ECFP by running an agarose gel which showed good results.  Expected  fragment lengths were received. The SAMS-peptide however was  too short to be  visible on the gel (67 bp).<br>
+
-
                                  <strong>Prepared for PCR 1-5. </strong>The miniprepped ECFP and EYFP  were  diluted to a stock concentration of 1 ng/ ml to use for the PCR.   The concentrations of the first PCR product  were measured using  nanodrop and then diluted to 1 ng/ml.<br>
+
-
                                  snf1: 20.5 ng/ml<br>
+
-
                                  snf4: 122.6 ng/ml<br>
+
-
                                  SAMS: 114.9 ng/ml<br>
+
-
                                </p>
+
-
                                <p> Karl and  Adnan<br>
+
-
                                  We made  duplicates of the haploid Snf4∆ and wildtype strains on new  plates in case the  original plate would be contaminated. Prepared a  sporulation medium that was  autoclaved and poured into empty growth  plates to stay in room temperature over  night.</p>
+
-
                                  <br></li>
+
-
        <li>  <p> <strong>2010-07-12</strong><br>
+
-
                                  Katarina  and Julia<br>
+
-
                                    <strong>Amplification of genomic SNF1 DNA by PCR. </strong>All  primers were resuspended in  mq-water according to the volume on the  lable, the final concentration 100 pg/ml. The primers were then diluted  10x  and the SAMS-peptide templates were diluted 1000x. PCR reactions  were run  amplifying subunits Snf1 and Snf4 and also the SAMS-peptide.  YPD medium and  agar plates were prepared (yeast).<br>
+
-
                                  </p>
+
-
                                  <p> Adnan and  Kemal<br>
+
-
                                <strong>Miniprep purification </strong>of EYFP and ECFP plasmides from  overnight culture. The concentrations were measured using Nanodrop:<br>
+
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                                  ECFP1: 99 ng/ml<br>
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                                  ECFP2: 89.9<br>
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                                  EYFP1: 104, 7 <br>
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                                  EYFP2: 172,5<br>
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                                  The  concentrations were verified with  electrophoresis. The plasmids were cut with  restriction enzymes EcoRI  and Pst1.<br>
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                                  ECFP, plasmid pSB1AK3, fragment sizes 750 bp and 3200 bp.<br>
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                                  EYFP, plasmid pSB1A2, fragment sizes 750 bp and 2050 bp.</p></li>
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                                <br>
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                                <li><p><strong>2010-07-11</strong><br>
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                                    Katarina<br>
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                                    Made  overnight cultures with colony 3  and 4 plasmid (not single cultures) and twp  each of EYFP and ECFP.
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                                    </p>
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                                </li>
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                                <br>
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                                <li><p><strong>2010-07-10</strong><br>
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                                    Julia<br>
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                                    Moved  plated bacteria from incubator to refrigerator. <br>
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                                  </p>
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                                </li>
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                                <li>
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                                    <p><strong>2010-07-09</strong><br>
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                                      Malin and  Julia<br>
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                                      The SOC  medium was autoclaved.  Competent E.coli cells were transformed with our FPs  through heat shock  and plated on petri dishes. 20 new LB-amp plates were made.<br>
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                                      Adnan and  Katarina<br>
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                                      Picked up  primers at Chalmers and  checked if they were ok. The concentration of the  genomic DNA (SNF1  gene) were measured. Prepared a master mix so that the  PCR-reactions  could be started Monday morning.</p>
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                                  <br>
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                                </li>
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                                <li> 
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                                  <p><strong>2010-07-08</strong><br>
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                                    Malin,  Julia and Peidi<br>
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                                    The two  BioBricks EYFP and ECFP were  extracted from the kit plates and stored in the  freezer. A SOC-medium  was prepared.<br>
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                                  </p>
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                                </li>
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                                <li>
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                                  <p><strong>2010-07-07 </strong><br>
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                                    Adnan,  Katarina and Julia<br>
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                                    As the gels  from Tuesday showed  ambigous results we decided to redo the miniprep of the  plamids. We  used the overnight preparations of the two unused colonies (3 &amp;  4).  The miniprep went smoothly except for major difficulties regarding    untrustworthy pipettes. The pipettes marked with green tape are the ones  to use  from now on together with the tips with the same markings! A  gel was molded and  this time we decided to use a full gel to get a  better resolution.  The results on the gel were very good.  The plasmids  had clearly been cut in one or  two places and the fragment sizes seem  to correspond to the expected sizes.<br>
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                                  </p>
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                                </li>
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                                <li> 
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                                  <p><strong>2010-07-06</strong><br>
+
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                                    Malin, Adnan and Peidi<br>
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                                    Colony 1 and 2 were picked to do a  miniprep.  The plasmids were extracted and a plasmid integrity check was  performed. By  cutting with BamHI, one cut were made to get the size of  the plasmid. Then we  cut with ClaI which is a two-cutter that gives a  fragment that is 600 bp and  one that is 6800. The gel which should  confirm this was not satisfactory since  the 600 band was missing.<br>
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                                  </p>
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                                </li>
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                                <li>
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                                  <p><strong>2010-07-05</strong><br>
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                                    Malin and Katarina<br>
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                                    An overnight culture were made from 4  different  colonies of E.coli containing the plasmid pSB-GM1.</p>
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                                </li>
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                        <p> <a href="#">Home</a> | <a href="#"><abbr title="Chalmers University of Technology">CTH</abbr></a> | <a href="#">Sponsors</a> | <a href="#">About Us</a> | <a href="#">Contact Us</a></p> <p> Chalmers University of Technology, Gothenburg, SWEDEN </p>
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Latest revision as of 12:15, 1 October 2010

Chalmers University of Technology

Team
Introduction CHALMERS Supervisors Students
FUSS
What's all about? Background Protein Fusion Methods Preliminary Results

CHALMERS UNIVERSITY OF TECHNOLOGY, GOTHENBURG

We are a team of 8 students from Chalmers University of Technology who will represent Gothenburg, SWEDEN in this year’s iGEM competition. We have started with a promising idea that combines cutting edge technologies available in the field of Synthetic Biology. Our research basically aims to constructing an optical reporter mechanism for cellular stress by tagging the stress activated SNF1 complex in yeast with fluorescent markers.

The project is performed with two main experimental pathways: Both experimental setups will utilize FRET to visualize the conformational change that is the result SNF1 activation. The first approach consists of fusing fluorescent two proteins, EYFP and ECFP, with the SNF1 complex. The principal is that when the protein is activated it undergoes a conformational change and this should be indicated by a change in the FRET-signal. The second approach utilizes a SAMS-peptide with fluorescent proteins fused to each end. The SAMS-peptide will be phosphorylated by the active SNF1-complex and undergo a conformational change that again will be indicated by the FRET-signal.

The long term ambition of this project it is to use the results in the pharmaceutical industry when performing high-throughput screening for new substances or finding the correct drug concentrations to use. The yeast cells with the modified SNF1-complex can be moved through a micro-fluidic system, gradually exposing them to an array of substances or a concentration gradient thereby easily finding at which concentration or by what substance the cells are stressed.

As of present we have purified the plasmid backbones that will be used together with fusion protein insert to transfect the yeast cells. The fusion protein primers has arrived and we will start working with the fusion PCR as of this week. We have completed the 3D models of the fusion proteins and the results look very promising, we will soon be able to present docking predictions with the complex.

Introduction

Who we are?
Left to right: Per Sunnerhagen, Markus Wilhelmsson, Malin Lindén, Karl Almén Burman, Adnan Kadic, Katarina Risö, Peidi Liu, Goutham Vemuri, Lokeshwaran Manoharan, Kemal Sanli, Julia Fransson

 

What we are doing?
Synthetic readout of cellular stress

The cellular stress is sensed by a key protein called AMP-activated protein kinase (AMPK). The AMPK protein complex is conserved among all eukaryotes, including yeast, plants and humans.

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Chalmers University of Technology, Gothenburg, SWEDEN

Retrieved from "http://2010.igem.org/Team:Gothenburg-Sweden/test"