Difference between revisions of "Team:Chalmers-Gothenburg/Results"

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{{Chalmers-Gothenburg}}
 
 
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 +
/*# sourceMappingURL=bootstrap.min.css.map */
 +
 +
</style>  <!------------------ bootstrap.min.css STOP ---------------------------------->
  
<h1>Results</h1>
+
<style type="text/css"> /*------------------ style_170803.css START ----------------------------------*/
  
<p>Here you can describe the results of your project and your future plans. </p>
 
  
<h5>What should this page contain?</h5>
+
@import url(https://fonts.googleapis.com/css?family=Rubik|Lato);
<ul>
+
<li> Clearly and objectively describe the results of your work.</li>
+
<li> Future plans for the project. </li>
+
<li> Considerations for replicating the experiments. </li>
+
</ul>
+
  
<h5>You should also describe what your results mean: </h5>
+
* {
 +
  margin:0;
 +
  padding:0;
 +
}
  
<ul>
+
body {
<li> Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for. </li>
+
  background-color: #e6e6e6;
<li> Show data, but remember all measurement and characterization data must be on part pages in the Registry. </li>
+
<li> Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project. </li>
+
  font-family:'Lato', sans-serif;
</ul>
+
}
  
</div>
+
/*----------------Overriding dumb wiki stuff----------------------------------*/
  
<div class="clear"></div>
+
#content {
 +
    width: 100%;
 +
    padding: 0;
 +
    margin-left: 0;
 +
    margin-top: -12px; /*Couldn't figure out why we got a white bar above all content, so I cheated a bit*/
 +
}
  
<div class="column half_size" >
 
  
 +
/*This changes margins that messed with the header navbar*/
 +
.mw-content-ltr ul, .mw-content-rtl .mw-content-ltr ul {
 +
    margin: 0;
 +
}
  
<h5> Project Achievements </h5>
+
/*This is the forced heading and logo. Why? :( */
 +
#top_title {
 +
    display: none;
 +
}
  
<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
+
/*Something caused the sideMenu to start out displayed instead of hidden. This fixes that.*/
 +
#sideMenu {
 +
    display: none;
 +
}
  
<ul>
+
/*---------------Index page header (above header nav bar)------------------------------*/
<li>A list of linked bullet points of the successful results during your project</li>
+
<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
+
</ul>
+
  
</div>
+
/*This is for the part above the header navbar with the title and logo*/
 +
#header_navbar_top {
 +
  background-color: #000000;
 +
  background-image: url("pictures_17/backgrounds/korsvagen_5.png"); /*linear-gradient(to bottom, #7bac78, #016243);*/ 
 +
background-repeat: no-repeat;
 +
  background-position: center;
 +
background-size: 100% 100%; /*60% 100%*/
 +
/*border: 3px solid #000000;*/
 +
text-align: left;
 +
position:top;
 +
  min-height: 750px;
 +
height:auto;
 +
background-attachment: fixed;
 +
  background-position: center;
 +
  background-repeat: no-repeat;
 +
  background-size: cover;
  
 +
  /*padding-right: 10px; /*100--> 250*/
 +
  /*padding-left: 10px; /*250--> 250px*/
 +
 +
/*height: 270px;/*220px
 +
  min-width: 70%; /*minimum width of header. Don't change from 1200px, title fucks up
 +
  min-height: 70%; /*don't think does anything
 +
margin-left: auto; /*The width of the header. Keep on auto to avoid gray edges
 +
  margin-right: auto; /* --"--
 +
  padding: 40px;/*Find another way to move the logo down a bit?*/
 +
  border-bottom: 5px solid transparent; /*A lot of code for the line between the header and the body */
 +
  -moz-border-image: -moz-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%);*/
 +
  -webkit-border-image: -webkit-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%)*/
 +
  border-image: linear-gradient(to right, #000000 0%, #404040, #000000 100%); /*(to right, #016243 0%, #b5deca, #016243 100%);*/
 +
  border-image-slice: 1;
 +
  -webkit-touch-callout: none;
 +
  -webkit-user-select: none;
 +
  -khtml-user-select: none;
 +
  -moz-user-select: none;
 +
  -ms-user-select: none;
 +
  user-select: none;
 +
  cursor: default;
  
<div class="column half_size" >
+
}
  
<h5>Inspiration</h5>
+
/*This is the title of the project*/
<p>See how other teams presented their results.</p>
+
#header_navbar_top_title {
<ul>
+
/*Sizes in px*/
<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
+
display:inline-block;
<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
+
padding-top:650px;
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
+
  padding-right:20%;
</ul>
+
  font-size: 100px;
 +
  line-height: 45px;
 +
  color: #c91a03;
 +
text-shadow: 3px 3px #000000;
 +
  font-family:'Lato', cursive; /*Dancing Script, Marck Script (meh), Tangerine (ok), Sacramento (meh), Allura (nej), Parisienne (meh), Montez(meh)*/
 +
font-weight: bold;
 +
text-align:left;
 +
margin-left:10%;
 +
 +
/*margin-right:10px; */
 +
/*For the title on index*/
 +
/*background-color: transparent;
 +
  font-size: 60pt; /*35pt
 +
font-family:"Lato", cursive; /*Dancing Script, Marck Script, Tangerine, Sacramento, Allura, Parisienne, Montez
 +
  color:#c91a03;
 +
line-height: 45pt;
 +
  padding-top: 35%;
 +
font-weight: bold;
 +
text-align:left;
 +
margin-left:200px;*/
 +
}
  
</div>
+
#header_navbar_top_subtitle {
 +
  color:#c91a03;
 +
  font-size: 40pt;
 +
line-height: 30pt;
 +
  padding-top: 1%;
 +
  padding-bottom: 20px;
 +
font-family:"Lato", cursive;
 +
text-align:left;
 +
margin-left:10%;
 +
margin-right:25%;
 +
text-shadow: 3px 3px #000000;
 +
}
  
 +
.subheader{
 +
color:#808080; /*#7e1002;*/
 +
text-align:center;
 +
font-size:50pt;
 +
padding-top:150px;
 +
padding-bottom:200px;
 +
}
 +
 +
 +
#header_navbar_top_logo {
 +
  display:inline-block;
 +
position: absolute;
 +
  width:9vw; /*20% 20%*/
 +
    height:9vw;
 +
    right:10%;
 +
    bottom:12%;
 +
 +
}
 +
 +
.overview {
 +
padding-bottom:100px;
 +
}
 +
 +
/*logo in the right corner*/
 +
.logo_pos_right{
 +
position: fixed;
 +
/*position in px*/ 
 +
/*  width:20px;
 +
    height:20px;
 +
    right:150px;
 +
    bottom:150px;*/
 +
/*position in percent*/ 
 +
  width:7vw; /*20% 20%*/
 +
    height:7vw;
 +
    right:3%;
 +
    bottom:5%;
 +
}
 +
 +
 +
#header_navbar_top_logo {
 +
  display:inline-block;
 +
}
 +
 +
 +
/*--------------------Header navbar--------------------------------------------*/
 +
 +
 +
/*body {
 +
    font-family: helvetica;
 +
margin:0;
 +
}*/
 +
 +
/*The wrapper and the nav bar have to be the same height, or the page will jump when the nav bar sticks.*/
 +
#header_navbar_wrapper, .header_navbar_ul {
 +
    height: 70px;
 +
}
 +
 +
.header_navbar_ul {
 +
  list-style-type: none; /*No bullet points*/
 +
  margin: 0;
 +
  padding: 0;
 +
  background-repeat: no-repeat;
 +
  background-color: #c91a03;
 +
border-bottom: 2px solid black;
 +
 +
top: 0;
 +
  width: 100%;
 +
  min-width: 1440px;
 +
  z-index:9999;
 +
  -webkit-touch-callout: none;
 +
  -webkit-user-select: none;
 +
  -khtml-user-select: none;
 +
  -moz-user-select: none;
 +
  -ms-user-select: none;
 +
  user-select: none;
 +
}
 +
 +
/*Having the header navbar stick to the top of the screen after it reaches that position*/
 +
.header-nav_stick {
 +
    position: fixed;
 +
    width: 100%;
 +
    top: 16px;
 +
    z-index: 9999;
 +
}
 +
 +
/*Current page header highlight*/
 +
.home #home_link,
 +
.team #team_link,
 +
.achievements #achievements_link,
 +
.HP #HP_link,
 +
.modelling #modelling_link,
 +
.notebook #notebook_link,
 +
.parts #parts_link,
 +
.project #project_link,
 +
.safety #safety_link
 +
{
 +
    color: white;
 +
text-decoration:underline;
 +
 +
}
 +
 +
 +
.dropbtn {
 +
    background-color: transparent;
 +
    color: white;
 +
    padding: 8px;
 +
    /*font-size: 16px;*/
 +
    border: none;
 +
    cursor: pointer;
 +
}
 +
 +
.dropdown,
 +
#header_navbar_home {
 +
    position: relative;
 +
    display: inline-block;
 +
    z-index:9999;
 +
    font-family:'Lato', sans-serif;
 +
}
 +
 +
.dropdown a,
 +
#header_navbar_home a {
 +
  display: block; /*Displaying the links as block elements makes the whole link
 +
  area clickable (not just the text), and it allows us to specify padding (and
 +
  height, width, margins, etc. if you want)*/
 +
  color: #e6e6e6;/*b5deca*/
 +
  text-align: left;
 +
  text-decoration: bold;
 +
  padding: 8px; /*Since block elements take up the full width available, they
 +
  cannot float next to each other. Therefore, specify some padding to make them look good*/
 +
  font-size: 14pt;
 +
  font-weight:bold;
 +
  line-height: 54px;
 +
}
 +
 +
 +
/*Size settings for the dropdown menu content*/
 +
.dropdown-content a {
 +
    line-height: 30px;
 +
    font-size: 12pt;
 +
}
 +
 +
/* Change the link color on hover over either dropdown button, or the dropdown menu items. Also, rounded top borders */
 +
.header_navbar_ul li:hover > a {
 +
    background-color: #7e1002; /*#7e1002*/
 +
    color: white;
 +
    border-radius: 10px 10px 0 0;
 +
text-decoration:none;
 +
 +
}
 +
 +
 +
#header_navbar_home a:hover {
 +
    color: white;
 +
    border-radius: 10px 10px 0 0;
 +
border-bottom: 2px solid black;
 +
}
 +
 +
 +
/*Top right rounded highlight for first list element in dropdown menu*/
 +
.dropdown-content a:first-child:hover {
 +
    border-radius: 0 10px 0 0;
 +
}
 +
 +
/*bottom rounded highlights for last list element in dropdown menu*/
 +
.dropdown-content a:last-child:hover {
 +
    border-radius: 0 0 10px 10px;
 +
}
 +
 +
.dropdown-content {
 +
    display: none;
 +
    position: absolute;
 +
    background-color: #c91a03;
 +
    box-shadow: 0px 8px 16px 0px rgba(0,0,0,0.2);
 +
    width: auto !important; /*to avoid line breaks*/
 +
    white-space: nowrap; /*to avoid line breaks*/
 +
    min-width: 200px;
 +
    border-radius: 0 10px 10px 10px;
 +
}
 +
 +
.dropdown-content a {
 +
    color: white;
 +
    padding: 12px 16px;
 +
    text-decoration: none;
 +
    font-weight: bold;
 +
    display: block;
 +
}
 +
 +
.dropdown-content a:hover {
 +
    background-color: #7e1002;/*#7e1002*/
 +
    /*border-radius: 10px 10px 10px 10px; /*Temprorary solution. I'd prefer to only round the corners that touch the corners of the menu*/
 +
    color: white;
 +
}
 +
 +
.dropdown:hover .dropdown-content {
 +
    display: block;
 +
}
 +
 +
/*Styling for <hr> between menu items in the dropdown menus*/
 +
.header_navbar_ul hr {
 +
    border: 0;
 +
    height: 1px;
 +
    background-image: linear-gradient(to right,  rgba(0, 0, 0, 0), #ccc,  rgba(0, 0, 0, 0));
 +
    margin-top: 0;
 +
    margin-bottom: 0;
 +
}
 +
 +
/*I don't think this one does anything. Probably because of "".dropdown-content a:hover" covering it.*/
 +
.dropdown:hover .dropbtn {
 +
    background-color: #111;
 +
}
 +
 +
 +
 +
/*---------------------------Footer with sponsor logos----------------------------*/
 +
 +
footer hr  {
 +
    border: 0;
 +
    height: 0;
 +
    /*border-top: 1px solid rgba(0, 0, 0, 0.1);*/
 +
    border-bottom: 1px solid rgba(255, 255, 255, 0.3);
 +
    width: 80%;
 +
    text-align: center;
 +
    margin-left: 10%;
 +
    margin-top: 50px;
 +
}
 +
 +
#footer-contact-row {
 +
  padding-bottom: 50px;
 +
}
 +
 +
#footer-contact-row a {
 +
  color: #999999;
 +
  text-decoration: none;
 +
  padding-left: 15px;
 +
  padding-right: 15px;
 +
}
 +
 +
#footer-contact-row a:hover {
 +
  color: #c91a03;
 +
}
 +
 +
#footer-contact-row hr  {
 +
    border: 0;
 +
    height: 0;
 +
    /*border-top: 1px solid rgba(0, 0, 0, 0.1);*/
 +
    border-bottom: 1px solid rgba(255, 255, 255, 0.3);
 +
    width: 30%;
 +
    text-align: center;
 +
    margin-left: 35%;
 +
}
 +
 +
#logorow {
 +
  width: 80%;
 +
  padding: 0;
 +
  margin: 0 auto 0 auto;
 +
}
 +
 +
.footerimg {
 +
  max-height: 100px;
 +
  max-width: 250px;
 +
}
 +
 +
.footerlogo {
 +
  display: inline-block;
 +
  vertical-align: bottom;
 +
  padding: 1em 1.5em 1em 1.5em;
 +
}
 +
 +
.footerlogo .on {
 +
  display: inline-block;
 +
}
 +
 +
.footerlogo:hover .on, .footerlogo .off {
 +
  display: none;
 +
}
 +
 +
.footerlogo:hover .off {
 +
  display: inline-block;
 +
}
 +
 +
/*---------------------------Center page styling---------------------------------*/
 +
 +
/*The wrap class is used for <div> in order to define a space where the
 +
side navigation bar and the page content (text, images etc) is kept*/
 +
 +
 +
.wrap {
 +
  /*width: 100%; /*auto--> 50%
 +
  text-align: center;
 +
  margin: auto; /*auto--> %
 +
  margin-top: 0px; /*40px --> 0px */
 +
 +
width: cover;
 +
  min-width:1440px;
 +
  text-align: center;
 +
  margin: auto;
 +
  margin-top: 0px;
 +
 +
background:#e6e6e6; 
 +
}
 +
 +
/*Attempt to wrap header*/
 +
.wrap-header {
 +
padding-right: auto; /*100--> 250*/
 +
  padding-left: auto; /*250--> 250px*/
 +
background-color: #e6e6e6;
 +
}
 +
 +
/*This is the content of the page, i.e. text images etc. Fits inside <div> with the wrap class*/
 +
.wrap-content {
 +
/*margin-top:75px;
 +
  padding-right: 25%; /*100--> 450*/ /*25%
 +
  padding-left: 25%; /*250--> 450px*/ /*25%
 +
background-color: transparent;
 +
border-style:dashed;*/
 +
 +
margin-top:100px;
 +
padding-right: 22%;
 +
  padding-left: 22%;
 +
background-color: transparent;
 +
 +
/*background-size:50px;*/
 +
}
 +
 +
/*Like .wrap-content, except with even margins, for pages where a side navigation bar is superfluous*/
 +
.wrap-content-no-side-nav {
 +
  padding-right: 75px;/*50px*/
 +
  padding-left: 75px;
 +
}
 +
 +
.wrap-content, .wrap-content-no-side-nav {
 +
  padding-top: 25px;
 +
}
 +
 +
.dashed_line_left_first {
 +
background-color:transparent;
 +
 +
border-style: none none dashed dashed;
 +
border-width:5px;
 +
border-color:#7e1002;
 +
padding-left:50px;
 +
padding-right:50px;
 +
padding-bottom:50px;
 +
padding-top:0px;
 +
}
 +
 +
.dashed_line_left {
 +
background-color:transparent;
 +
 +
border-style: none none dashed dashed;
 +
border-width:5px;
 +
border-color:#7e1002;
 +
padding:50px;
 +
}
 +
 +
.dashed_line_right {
 +
background-color:transparent;
 +
border-style: none dashed dashed none;
 +
border-width:5px;
 +
border-color:#7e1002;
 +
padding:50px;
 +
}
 +
 +
.dashed_line_right_last { /*No bottom border, only left*/
 +
background-color:transparent;
 +
border-style: none dashed none none;
 +
border-width:5px;
 +
border-color:#7e1002;
 +
padding:50px;
 +
}
 +
 +
.dashed_line_left_last { /*No bottom border, only right*/
 +
background-color:transparent;
 +
border-style: none none none dashed;
 +
border-width:5px;
 +
border-color:#7e1002;
 +
padding:50px;
 +
}
 +
 +
/*----------------------------Side navigation bar-------------------------------*/
 +
 +
#side-nav {
 +
    width: 25%;
 +
}
 +
 +
#side-nav nav {
 +
    /*width: 225px;
 +
    padding-left: 25px;
 +
    padding-top:80px;
 +
    float: left;
 +
border-style:dashed;*/
 +
 +
  width: 175px;
 +
    margin-left: 25px;
 +
    margin-top:140px;
 +
    float: left;
 +
border-style:solid;
 +
border-width:2px;
 +
border-color:#000000;
 +
 +
}
 +
 +
#side-nav a {
 +
    color: #333;
 +
    display: block;
 +
    padding: 10px;
 +
    text-align: left;
 +
    text-decoration: none;
 +
background: #f2f2f2;
 +
font-size:12pt !important;
 +
}
 +
 +
#side-nav a.active {
 +
    background: #c91a03;
 +
    color: #1a1a1a;
 +
font-size:12pt !important;
 +
}
 +
 +
#side-nav .sub-level {
 +
    padding-left: 30px;
 +
    /*font-size: 12pt;*/
 +
}
 +
 +
#side-nav a:hover {
 +
  background: #ffffff; /*Before: #dcdbdb;*/
 +
  color: black;
 +
font-size:12pt !important;
 +
font-weight:bold;
 +
text-decoration:underline;
 +
text-decoration-color:#c91a03;
 +
}
 +
 +
 +
.side-nav_stick {
 +
    position: fixed;
 +
    top: 0;
 +
    z-index: 0;
 +
    margin-top:120px;
 +
}
 +
 +
.hidden-sidenav {
 +
  opacity: 0;
 +
}
 +
 +
#side-nav nav {
 +
-webkit-transition: opacity 200ms ease-in-out;
 +
    -moz-transition: opacity 200ms ease-in-out;
 +
    -ms-transition: opacity 200ms ease-in-out;
 +
    -o-transition: opacity 200ms ease-in-out;
 +
}
 +
 +
 +
/*---------------------------Profile card styling-------------------------------------*/
 +
.profile-table {
 +
  margin: 0 auto 0 auto;
 +
  border-spacing: 30px;
 +
  border: 0 !important;
 +
}
 +
 +
.profile-table td {
 +
  border: 0 !important;
 +
}
 +
 +
.profile-main {
 +
  height: 350px;
 +
  width: 300px;
 +
  position: relative;
 +
  margin: 10px;
 +
}
 +
 +
.profile-container {
 +
  height: 350px;
 +
  width: 300px;
 +
  position: absolute;
 +
  display: block;
 +
  top: 0;
 +
  left: 0;
 +
-webkit-border-radius: 5px;
 +
-moz-border-radius: 5px;
 +
border-radius: 5px;
 +
box-shadow: 1px 1px 4px #666;
 +
padding: 1%;
 +
background: #fffef9;
 +
background: rgb(252,255,244); /* Old browsers */
 +
background: -moz-linear-gradient(top,  #fffffb 0%, #fffef9 100%); /* FF3.6+ */
 +
background: -webkit-gradient(linear, left top, left bottom, color-stop(0%,rgba(252,255,244,1)), color-stop(100%,rgba(219,218,201,1))); /* Chrome,Safari4+ */
 +
background: -webkit-linear-gradient(top,  #fffffb 0%,#fffef9 100%); /* Chrome10+,Safari5.1+ */
 +
background: -o-linear-gradient(top,  #fffffb 0%,#fffef9 100%); /* Opera 11.10+ */
 +
background: -ms-linear-gradient(top,  #fffffb 0%,#fffef9 100%); /* IE10+ */
 +
background: linear-gradient(top,  #fffffb 0%,#fffef9 100%); /* W3C */
 +
filter: progid:DXImageTransform.Microsoft.gradient( startColorstr='#fcfff4', endColorstr='#dbdac9',GradientType=0 ); /* IE6-9 */
 +
}
 +
 +
.profile-image {
 +
  margin: 15px auto 0 auto;
 +
  height: 250px;
 +
  width: 250px;
 +
  position:relative;
 +
}
 +
 +
.profile-content {
 +
  display: none;
 +
  width: 300px;
 +
  margin: 70px auto 0 auto;
 +
  text-align: justify;
 +
}
 +
 +
.profile-container:hover .profile-content {
 +
  display: block;
 +
}
 +
 +
.profile-container:hover .profile-image {
 +
  position: relative;
 +
}
 +
 +
.profile-name {
 +
  padding-top: 15px;
 +
  color: #c91a03;
 +
  font-weight: bold;
 +
  font-size: 15pt;
 +
}
 +
 +
.profiletext {
 +
  font-size: 10pt !important;
 +
  margin-top: 10px;
 +
  line-height: 150%;
 +
  text-align: justify;
 +
}
 +
 +
.profilejob {
 +
  font-size:12pt !important;
 +
}
 +
 +
/*-------------------------------GENERAL STUFF-------------------------------------*/
 +
 +
div {
 +
  text-align: center;
 +
}
 +
 +
.header_test {
 +
background-position: top;
 +
background-attachment: fixed;
 +
background-repeat: no-repeat;
 +
-webkit-background-size: cover;
 +
-moz-background-size: cover;
 +
-o-background-size: cover;
 +
background-size: cover;
 +
        padding-top: 100vh;
 +
        background-image: url("https://static.igem.org/mediawiki/2017/0/00/T--Chalmers-Gothenburg--Achievements_Results.jpg");
 +
 +
/*A lot of code for the line between the header and the body */
 +
border-bottom: 3px solid transparent;
 +
  -moz-border-image: -moz-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%);*/
 +
  -webkit-border-image: -webkit-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%)*/
 +
  border-image: linear-gradient(to right, #000000 0%, #404040, #000000 100%); /*(to right, #016243 0%, #b5deca, #016243 100%);*/
 +
  border-image-slice: 1;
 +
  -webkit-touch-callout: none;
 +
  -webkit-user-select: none;
 +
  -khtml-user-select: none;
 +
  -moz-user-select: none;
 +
  -ms-user-select: none;
 +
  user-select: none;
 +
  cursor: default;
 +
}
 +
 +
 +
/* Banner: name of the page */
 +
#banner {
 +
height: 100%;
 +
        position: relative;
 +
        bottom: 50px;
 +
    float: left;
 +
    width: 100%;
 +
        font-size: 80pt; /*35pt*/
 +
font-family:"Lato", cursive;
 +
        color:#c91a03 ;
 +
line-height: 65pt;
 +
        text-shadow: 5px 5px #000000;
 +
}
 +
 +
#banner h3 {
 +
display: block;
 +
position: absolute;
 +
bottom: 50px;
 +
left: 200px;
 +
        font-size: 80pt; /*35pt*/
 +
font-family:"Lato", cursive;
 +
        color:#c91a03 ;
 +
line-height: 65pt;
 +
        text-shadow: 5px 5px #000000;
 +
        /*border-style:dashed;*/
 +
padding: 20px 20px 20px 20px;
 +
 +
}
 +
 +
#banner h4 {
 +
display: block;
 +
position: absolute;
 +
bottom: -40px;
 +
left: 200px;
 +
        font-size: 40pt; /*35pt*/
 +
font-family:"Lato", cursive;
 +
        color:#c91a03 ;
 +
line-height: 65pt;
 +
        text-shadow: 3px 3px #000000;
 +
        /*border-style:dashed;*/
 +
padding: 20px 20px 20px 20px;
 +
}
 +
 +
 +
.header-text {
 +
  background-color: transparent;
 +
  font-size: 80pt; /*35pt*/
 +
font-family:"Lato", cursive; /*Dancing Script, Marck Script, Tangerine, Sacramento, Allura, Parisienne, Montez*/
 +
  color:#c91a03 ;/*Alternatives: #c91a03, #ff0000, #fd4833, #fb2104*/
 +
line-height: 65pt;
 +
  padding-top: 30%;
 +
font-weight: bold;
 +
text-align:left;
 +
margin-left:10%;
 +
text-shadow: 5px 5px #000000;
 +
/*border-style:dashed;
 +
border-color:red;*/
 +
}
 +
 +
.header-subtitle {
 +
  color:#c91a03;
 +
  font-size: 40pt;
 +
line-height: 15pt;
 +
  padding-top: 2%;
 +
  padding-bottom: 20px;
 +
font-family:"Lato", cursive; /*Dancing Script, Marck Script, Tangerine, Sacramento, Allura, Parisienne, Montez*/
 +
text-align:left;
 +
margin-left:10%;
 +
text-shadow: 2px 2px #000000;
 +
}
 +
 +
 +
/*--------------Basic text and content styling---------------------------------*/
 +
p {
 +
  font-size: 12pt !important;
 +
  text-align: justify;
 +
  text-justify: inter-word;
 +
  /*text-indent: 20px;*/
 +
  line-height: 200%;
 +
  margin-bottom: 10px;
 +
color:#1a1a1a;
 +
}
 +
 +
h2 {
 +
text-align:left;
 +
}
 +
 +
.h2style {
 +
color:#1a1a1a;
 +
font-size:25pt;
 +
text-align:center;
 +
}
 +
 +
h3 {
 +
  text-align: left;
 +
 
 +
}
 +
 +
figcaption {
 +
  color: black;
 +
  font-size: 11pt;
 +
  text-align: justify;
 +
  padding-left: 80px;
 +
margin-left:50px;
 +
 
 +
}
 +
 +
figcaption b {
 +
  color: #c91a03;
 +
}
 +
 +
.subtitle {
 +
 +
color:#000000;
 +
font-size:30pt !important;
 +
text-align:center;
 +
 +
border-bottom: 2px solid transparent; /*A lot of code for the line between the header and the body */
 +
  -moz-border-image: -moz-linear-gradient(left, #1a1a1a 0%, #808080, #1a1a1a 100%); /*(left, #016243 0%, #b5deca, #016243 100%);*/
 +
  -webkit-border-image: -webkit-linear-gradient(left, #1a1a1a 0%, #808080, #1a1a1a 100%); /*(left, #016243 0%, #b5deca, #016243 100%)*/
 +
  border-image: linear-gradient(to right, #1a1a1a 0%, #808080, #1a1a1a 100%); /*(to right, #016243 0%, #b5deca, #016243 100%);*/
 +
  border-image-slice: 1;
 +
  -webkit-touch-callout: none;
 +
  -webkit-user-select: none;
 +
  -khtml-user-select: none;
 +
  -moz-user-select: none;
 +
  -ms-user-select: none;
 +
  user-select: none;
 +
  cursor: default;
 +
}
 +
 +
.list {
 +
  margin-left:50px;
 +
  list-style-position: outside;
 +
  text-align: justify;
 +
  text-justify: inter-word;
 +
  margin-top: 10px;
 +
  font-size: 12pt;
 +
  line-height: 125%;
 +
}
 +
 +
.sponsor-picture {
 +
display: inline-block;
 +
border-style:dashed;
 +
width: 500px;
 +
  height:170px;
 +
padding: 10px;
 +
}
 +
 +
.sponsor-picture2 {
 +
width: 200px;
 +
  margin-left: 50px;
 +
margin-bottom:10px;
 +
float:right;
 +
}
 +
 +
.sidetitle {
 +
 +
color:#1a1a1a;
 +
font-size:30pt;
 +
text-align:center;
 +
 +
border-bottom: 2px solid transparent; /*A lot of code for the line between the header and the body */
 +
  -moz-border-image: -moz-linear-gradient(left, #000000 0%, #808080, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%);*/
 +
  -webkit-border-image: -webkit-linear-gradient(left, #000000 0%, #808080, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%)*/
 +
  border-image: linear-gradient(to right, #000000 0%, #808080, #000000 100%); /*(to right, #016243 0%, #b5deca, #016243 100%);*/
 +
  border-image-slice: 1;
 +
  -webkit-touch-callout: none;
 +
  -webkit-user-select: none;
 +
  -khtml-user-select: none;
 +
  -moz-user-select: none;
 +
  -ms-user-select: none;
 +
  user-select: none;
 +
  cursor: default;
 +
 +
}
 +
 +
.figure-caption {
 +
    display: table-caption;
 +
    caption-side: bottom; 
 +
}
 +
 +
.table-caption {
 +
    display: table-caption;
 +
    caption-side: top;   
 +
  color: black;
 +
  font-size: 11pt;
 +
  text-align: justify;
 +
  padding-left: 0px;
 +
  padding-right:0px;
 +
margin-left:0px;
 +
margin-right:0px;
 +
}
 +
 +
th {
 +
    text-align: center;
 +
}
 +
 +
figure {
 +
  display:table;
 +
  text-align:center;
 +
  margin: auto;
 +
  margin-bottom: 25px;
 +
  margin-top: 25px;
 +
}
 +
 +
/*---Reference List---*/
 +
 +
.reference_list {
 +
    text-align: left;
 +
    font-size:10pt; !important
 +
}
 +
 +
.reference_list ul {
 +
    list-style-type: none;
 +
    list-style-image: none;
 +
}
 +
 +
.reference_list li {
 +
    padding-bottom: 10px;
 +
}
 +
 +
/*--------------Links in text and lists are red when hovered------------------*/
 +
 +
 +
 +
p a:link {
 +
  text-decoration: none;
 +
  color: #c91a03;
 +
 
 +
}
 +
 +
p a:link:hover {
 +
  color: #7e1002;
 +
}
 +
 +
p a:visited {
 +
  text-decoration: none;
 +
  color: #c91a03;
 +
}
 +
 +
 +
 +
td a:link {
 +
  text-decoration: none;
 +
  color: #c91a03;
 +
 
 +
}
 +
 +
td a:link:hover {
 +
  color: #7e1002;
 +
}
 +
 +
td a:visited {
 +
  text-decoration: none;
 +
  color: #c91a03;
 +
}
 +
 +
 +
h1 a:link {
 +
  font-weight: bold;
 +
color: #c91a03;
 +
}
 +
 +
h1 a:link:hover {
 +
  font-weight: bold;
 +
  color: #7e1002;
 +
}
 +
 +
h1 a:visited {
 +
  /*font-weight: bold;*/
 +
color:#c91a03;
 +
}
 +
 +
/*Commented before, want to know what it does*/
 +
.list a:link {
 +
  text-decoration: none;
 +
  color: #c91a03;
 +
}
 +
 +
.list a:visited {
 +
  text-decoration: none;
 +
  color: #c91a03;
 +
}
 +
 +
.list a:hover {
 +
  text-decoration: none;
 +
  color: #7e1002;
 +
}
 +
 +
.text a:link {
 +
  text-decoration: none;
 +
  color: #c91a03;
 +
}
 +
 +
.text a:visited {
 +
  text-decoration: none;
 +
  color: #c91a03;
 +
}
 +
 +
.text a:hover {
 +
  text-decoration: none;
 +
  color: #7e1002;
 +
}
 +
 +
.profiletext a:link {
 +
  text-decoration: none;
 +
  color: #c91a03;
 +
  font-weight: bold;
 +
}
 +
 +
.profiletext a:visited {
 +
  text-decoration: none;
 +
  color: #c91a03;
 +
  font-weight: bold;
 +
}
 +
 +
 +
.profiletext a:hover {
 +
  text-decoration: none;
 +
  color: #7e1002;
 +
}
 +
 +
/*superscript and subscript modification so it doesn't fuck up line-height in multiple lined text*/
 +
sub, sup {
 +
font-size: 75%;
 +
line-height: 0;
 +
position: relative;
 +
vertical-align: baseline;
 +
}
 +
sup {
 +
top: -0.5em;
 +
}
 +
sub {
 +
bottom: -0.25em;
 +
}
 +
 +
.thankyoutext {
 +
  font-size: 16pt;
 +
  text-align: justify;
 +
  text-justify: inter-word;
 +
  margin-top: 10px;
 +
  text-indent: 20px;
 +
  line-height: 150%;
 +
  font-weight: 500;
 +
}
 +
 +
 +
/*---------------------------BUTTON---------------------------------*/
 +
.button {
 +
  display: inline-block;
 +
  border-radius: 10px;
 +
  background-color: #5B9E00;
 +
  border: none;
 +
  color: #FFFFFF;
 +
  text-align: center;
 +
  font-size: 28px;
 +
  padding: 20px;
 +
  width: 250px;
 +
  transition: all 0.6s;
 +
  cursor: pointer;
 +
  margin: 5px;
 +
}
 +
 +
.button span {
 +
  cursor: pointer;
 +
  display: inline-block;
 +
  position: relative;
 +
  transition: 0.5s;
 +
}
 +
 +
.button span:after {
 +
  content: '»';
 +
  position: absolute;
 +
  opacity: 0;
 +
  top: 0;
 +
  right: -20px;
 +
  transition: 0.5s;
 +
}
 +
 +
.button:hover span {
 +
  padding-right: 25px;
 +
}
 +
 +
.button:hover span:after {
 +
  opacity: 1;
 +
  right: 0;
 +
}
 +
 +
/*-----------------------long button----------------------------------------*/
 +
.button_long {
 +
  display: inline-block;
 +
  border-radius: 10px;
 +
  background-color: #5B9E00;
 +
  border: none;
 +
  color: black;
 +
  text-align: left;
 +
  font-weight:600;
 +
  font-size: 15pt;
 +
  padding: 10px;
 +
  width: 100%;
 +
  transition: all 0.6s;
 +
  cursor: pointer;
 +
  margin-top: 10px;
 +
  box-shadow: 0 8px 16px 0 rgba(0,0,0,0.2), 0 6px 20px 0 rgba(0,0,0,0.19);
 +
}
 +
 +
.button_long span {
 +
  cursor: pointer;
 +
  display: inline-block;
 +
  position: relative;
 +
  transition: 0.5s;
 +
}
 +
 +
.button_long span:after {
 +
  content: '»';
 +
  position: absolute;
 +
  opacity: 0;
 +
  top: 0;
 +
  right: -20px;
 +
  transition: 0.5s;
 +
}
 +
 +
.button_long:hover span {
 +
  padding-right: 25px;
 +
}
 +
 +
.button_long:hover span:after {
 +
  opacity: 1;
 +
  right: 0;
 +
}
 +
 +
 +
/*-------------------------------------- NOTE!!!!!!!!!!!! Do not add the code for the slider. The buttons for previewing and save the wiki ------------------------------------------*/
 +
 +
</style> <!------------------ style_170803.css STOP ---------------------------------->
 +
 +
<!---------------------------------------- Insert jquery and script.js here, when you figure out how to make them work...------------------------------>
 +
 +
<!-------------------------------- jquery-3.0.0.min.js START ----------------------->
 +
<!-- WORKING!!!!!! -->
 +
<script type="application/javascript">
 +
 +
/*! jQuery v3.0.0 | (c) jQuery Foundation | jquery.org/license */
 +
!function(a,b){"use strict";"object"==typeof module&&"object"==typeof module.exports?module.exports=a.document?b(a,!0):function(a){if(!a.document)throw new Error("jQuery requires a window with a document");return b(a)}:b(a)}("undefined"!=typeof window?window:this,function(a,b){"use strict";var c=[],d=a.document,e=Object.getPrototypeOf,f=c.slice,g=c.concat,h=c.push,i=c.indexOf,j={},k=j.toString,l=j.hasOwnProperty,m=l.toString,n=m.call(Object),o={};function p(a,b){b=b||d;var c=b.createElement("script");c.text=a,b.head.appendChild(c).parentNode.removeChild(c)}var q="3.0.0",r=function(a,b){return new r.fn.init(a,b)},s=/^[\s\uFEFF\xA0]+|[\s\uFEFF\xA0]+$/g,t=/^-ms-/,u=/-([a-z])/g,v=function(a,b){return b.toUpperCase()};r.fn=r.prototype={jquery:q,constructor:r,length:0,toArray:function(){return f.call(this)},get:function(a){return null!=a?0>a?this[a+this.length]:this[a]:f.call(this)},pushStack:function(a){var b=r.merge(this.constructor(),a);return b.prevObject=this,b},each:function(a){return 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  <head>  <!------------------ HTML START ---------------------------------->
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            <a href="#overview" class="active">Overview</a>
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            <a href="#receptors">Receptors</a>
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            <a href="#pathway">Pathway</a>
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            <a href="#future">Future directions</a>
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            <a href="#references">References</a>
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        <div class="target" id="overview">
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        <div class="dashed_line_left_first">
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          <h4 class="subtitle">Overview</h4>
 +
        <p class="text">
 +
The lung cancer biosensor was divided into two major parts: <b>receptor integration</b> and <b>pathway</b>. The native membrane localized GPCRs STE2 and STE3 were substituted with the two heterologous GPCRs Olf1258 and Ri7 in two different yeast mating types. These two heterologous GPCRs were fused with GFP to test membrane localization. Finally, the functionality of the GPCRs was tested using the plasmid pGFP, were P<sub><i>FUS1</i></sub> is coupled to GFP. P<sub><i>FUS1</i></sub> is a mating gene promoter induced after GPCR induction. For testing the functional pathway, the loxP-P<sub><i>TEF</sub></i>-loxP switch was investigated. Before the P<sub><i>TEF</i></sub> promoter is turned, GFP is expressed. Therefore, the decrease in GFP expression was tracked after the turning of the promoter, indicating that the promoter switch was successful.
 +
        </p>
 +
          </div>
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        </div>
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        <div class="target" id="receptors">
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          <div class="dashed_line_right"> 
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            <h4 class="sidetitle">Receptors</h4>
 +
            <h2 class="h2style">Integration of Olfr1258 and Ri7</h2>
 +
        <p class="text">
 +
For the biosensor to work, modifications of the two yeast strains, a and α, needed to be performed. One major modification was the substitution of the native GPCRs, STE2 and STE3, with the two heterogenous olfactory receptors, Ri7 and Olfr1258. This was executed using a CRISPR system with Cas9 and gRNA specific to STE2 and STE3. For more information see <a href="https://2017.igem.org/Team:Chalmers-Gothenburg/Project/constructs" target="blank">Project, constructs</a>.       
 +
        </p>
 +
        <p class="text">
 +
By using colony PCR, the substitution/integration of the GPCRs could be validated, after transformation of sequences for GPCR and CRISPR system. The colony PCRs showed successful results for the incorporation of both of the GPCRs into the genome of the respective yeast strain. Olfr1258 has been incorporated into the strain CENPK_111-61A and Ri7 into CENPK_11-11C.       
 +
        </p>
 +
            </div>
 +
 +
 +
      <div class="dashed_line_left"> 
 +
            <h2 class="h2style">Membrane localization</h2>
 +
          <p class="text">
 +
To enable visualization of the localization of the receptors in the yeast cells, the yeast was transformed with a plasmid containing the GPCRs fused together with GFP. The green fluorescence could then easily enable detection of the GPCRs. For the yeast strain with Olfr1258, the membrane localization of the GPCR could be verified. In the leftmost picture in Figure 1, it can be seen that Olfr1258 is clearly located in the outer membrane of the cell. In the rightmost picture, it can be seen that these cells fluoresce in the nuclear membrane which is clearly shown when the cells in this image is dividing. This image thereby suggest that the GPCR is localized in all membranes.
 +
        </p>
 +
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2017/9/91/T--Chalmers-Gothenburg--Olfr_GFP.png" style="height:196px; width:404px;">
 +
<figcaption><b>Figure 1.</b>The yeast expressing Olfr1258 fused together with GFP. The image visualizes the membrane-localization of Olfr1258 due to the green fluorescence expressed at the edges of the yeast cells. </figcaption>
 +
</figure>
 +
 +
        <p class="text">
 +
For the yeast cells expressing Ri7 fused together with GFP it could be visualized that this GPCR is membrane-localized as well, see Figure 2.
 +
        </p>
 +
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2017/3/36/T--Chalmers-Gothenburg--Ri7_GFP.png " style="height:207px; width:262px;">
 +
<figcaption><b>Figure 2.</b>The yeast expressing Ri7 fused together with GFP. The image visualizes that Ri7 is membrane-localized due to the green fluorescence expressed at the edges of the yeast cell. </figcaption>
 +
</figure>
 +
        </div>
 +
 +
      <div class="dashed_line_right"> 
 +
            <h2 class="h2style">Lethality of VOCSs in unmodified strain</h2>
 +
          <p class="text">
 +
A lethality test of the VOCs in unmodified yeast strain was performed to be able to examine what range of concentrations the cells could handle and survive in. This test was crucial for the project since the concentration that could be used in the other test of the VOCs together with the modified strain needed to be in a concentration that was known not to affect the cell growth.
 +
        </p>
 +
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2017/b/b3/T--Chalmers-Gothenburg--Lethality_test_Butanone_graph_gray.png" style="height:373px; width:493px;">
 +
<figcaption><b>Figure 3.</b> The lethality test for butanone showing the OD of the yeast cells over approximately 5 hours after exposure to different concentrations of butanone and DMSO.</figcaption>
 +
</figure>
 +
 +
          <p class="text">
 +
This experiment was designed to expose the cells to different concentrations of respective VOC as well as different amounts of DMSO. The OD was measured after 2, 4 and 5,5 hours.
 +
        </p>
 +
          <p class="text">
 +
From the lethality test for butanone it could be seen that this range of concentrations, the butanone itself did not kill the cells. The cells that were exposed to 1 μM (1% DMSO), 10 μM (1% DMSO),1 mM (0.1% DMSO) and 10 mM (1% DMSO) did grow without being affected. However, the cells that were exposed to a higher concentration of DMSO had a clear decreased growth rate.
 +
        </p>
 +
 +
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2017/2/2a/T--Chalmers-Gothenburg--Lethality_test_n-octanal_graph_gray.png" style="height:344px; width:507px;">
 +
<figcaption><b>Figure 4.</b> The lethality test for n-octanal showing the OD over approximately 5 hours after exposure to different concentrations of octanal and DMSO.</figcaption>
 +
</figure>
 +
 +
          <p class="text">
 +
n-Octanal showed to have more negative impact on the lethality of the cells. The cells that were exposed to 10 μM (1% DMSO) and 1 μM (1% DMSO) had the highest survival. The cells that were exposed to 100 μM (10% DMSO), 10 mM (1% DMSO) and the DMSO control (10% DMSO) had a clear decreased growth rate. However, the 1 mM (0.1% DMSO) showed a weak growth and it was clearly visualized that these cells were affected.
 +
        </p>
 +
          <p class="text">
 +
From these two lethality test, it can be concluded that the concentration of DMSO is crucial for the survival of the cells and the concentration should be below 1%. The concentration range tested for butanone did not affect the cells. However, the concentration range tested for n-octanal had an effect on the cells and the concentration of octanal should be kept between 1 μM and 10 μM for the cells to stay unaffected.
 +
        </p>
 +
        </div>
 +
 +
      <div class="dashed_line_left"> 
 +
            <h2 class="h2style">BioLector</h2>
 +
          <p class="text">
 +
The GPCRs’ ability to recognize and bind the VOCs, initiate the MAPK pathway and thus activate the P<sub><i>FUS1</i></sub> could be evaluated with a BioLector. Yeast with Ri7 and Olfr1258 incorporated was induced with n-octanal and butanone, respectively. A plasmid with GFP, pGFP, under the control of P<sub><i>FUS1</i></sub> was incorporated and thus, GFP would be expressed as a positive output. 
 +
        </p>
 +
          <p class="text">
 +
The result for Olfr1258 shows a slightly higher GFP expression when the yeast is induced with butanone, see Figure 5. For Ri7 there is no clear result. The higher GFP expression for Olfr1258 indicates that the receptor is able to sense the butanone and initiate the MAPK pathway which in turn activates the P<sub><i>FUS1</i></sub> promoter. However, one need to consider the possibility that such differences might occur due to varying growth rates compared to the cells’ autofluorescence and further tests would be needed. If the further tests strengthen the results, it shows that the Olfr1258 works as it should.
 +
        </p>
 +
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2017/b/b2/T--Chalmers-Gothenburg--Olfr1258_graph_gray.png" style="height:390px; width:598px;">
 +
<figcaption><b>Figure 5.</b>The results from the BioLector of Olfr1258. The different results for respective concentrations are plotted with GFP emission per OD at the y-axis against time on the x-axis. </figcaption>
 +
</figure>
 +
            </div>
 +
      </div>
 +
 +
        <div class="target" id="pathway">
 +
          <div class="dashed_line_right"> 
 +
            <h4 class="sidetitle">Pathway</h4>
 +
            <h2 class="h2style">Cre-Cas9</h2>
 +
        <p class="text">
 +
The Cre-Cas9 construct was successfully assembled together into a plasmid. To make the P<sub><i>FUS1</i></sub> promoter activate, pheromones are needed since P<sub><i>FUS1</i></sub> activation is accomplished through the yeast pheromone pathway. Pheromones are produced through the mating mechanism of the yeast. The activation of P<sub><i>FUS1</i></sub> promoter will lead to expression of Cre-recombinase so the LoxP-sites would switch. The switch-system was designed so that it would be non-reversible and by flipping the promoter P<sub><i>TEF1</i></sub>, expression of the GFP would stop and Cas9 would be expressed instead.
 +
        </p>
 +
        <p class="text">
 +
The two different mating strains successfully mated which was done using selection and microscopic imaging. Further fluorescence microscopy images and colony-PCR runs were done to verify that the switch worked. The results came back negative. Sequencing of the Cre-Cas9 construct showed mutations in the Cre-recombinase part and might be the reason to why these experiments were unsuccessful.
 +
        </p>
 +
        <p class="text">
 +
To truly make sure that the flip worked and that the problem was not due to the deviation in the sequence of P<sub><i>CRE</i></sub> or that the P<sub><i>FUS1</i></sub> promoter is too weak to induce expression, another test was done by using an inducible Cre-recombinase plasmid. The activation of Cre-recombinase in the plasmid is accomplished by the presence of galactose. The cell culture was grown in a Delft medium, containing galactose, and observed under a fluorescence microscope during three days. Three replicates were used and all gave a similar result. Figure 6 shows the fluorescence measurements during these three days for one colony. The GFP expression is clearly decreased with the passage of time.
 +
        </p>
 +
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/parts/f/fc/Cre-test.png" style="height:942px; width:537px;">
 +
<figcaption><b>Figure 6.</b> A schematic view over the three days the cells were grown in galactose. The left figure show a microscopic image of the cells to give a sense of the amount of cells and the right figure show the fluorescence of these cells.</figcaption>
 +
</figure>
 +
          </div>
 +
          <div class="dashed_line_left"> 
 +
        <p class="text">
 +
A colony-PCR was also performed for this test, in order to confirm that the flip worked at sequence level and to make sure that the decrease in expression comes from the switch and not something else. The gel electrophoresis, Figure 2, shows that the flip appear to have succeeded in one of the replicates tested. The clear band at 1300 bp indicates that the flip worked. The upper run shows a gel electrophoresis with only two primers. Only the cells who succeeded to perform the flip will show a band. The lower run shows a gel electrophoresis with three primers. If the flip did not succeed, a band at 500 bp will appear as opposed to 1300 bp, if the flip worked. Two colony-PCRs were run, due to the the fact that the flip might not have a 100 % efficiency, and since the PCR would favour the shorter construct. In the lower gel, there are two band shown for the second replicate, which indicate that the efficiency was not 100 %. Notable is that the other two replicates showed no bands at all in either test, which means that the PCR did not work for them. There should at least be a band in the lower run regardless of the direction of the promoter.
 +
        </p>
 +
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/parts/f/fc/Colony-PCR_of_the_flip.png" style="height:338px; width:201px;">
 +
<figcaption><b>Figure 7.</b>The gel electrophoresis result for the colony-PCR. In the first well the control is loaded, and the other three show the three replicates. Same goes for the lower run. A band at 1300 bp indicate a success in the flipping of the promoter, and a band at 500 bp show a failure. The ladder used is GeneRuler 1kb.</figcaption>
 +
</figure>
 +
 +
            <h2 class="h2style">Cre-gRNA</h2>
 +
        <p class="text">
 +
No further tests was done on this pathway. Please check out <a href="https://2017.igem.org/Team:Chalmers-Gothenburg/Description" target="blank">Project, description</a> or check out the reference for more information [1].
 +
        </p>
 +
      </div>
 +
    </div>
 +
 +
        <div class="target" id="future">
 +
          <div class="dashed_line_right"> 
 +
            <h4 class="sidetitle">Future directions</h4>
 +
        <p class="text">For future directions of this project, there are some aspects to consider;
 +
firstly, to make all parts of the system work and get a complete functional system, from the sensing of the VOCs to the output of <i>ADE2</i>.
 +
        </p>
 +
        <p class="text">
 +
Once a functioning system is finalized, several steps are required to allow it to be taken into use. To get the biosensor approved for usage in a screening program, extensive testing of the method is required both on healthy people and lung cancer patients. This to be able to show that the system can distinguish between healthy and sick patients.
 +
        </p>
 +
          </div>
 +
        </div>
 +
 +
        <div class="target" id="references">
 +
          <div class="dashed_line_left_last"> 
 +
            <h4 class="sidetitle">References</h4>
 +
            <div class="reference_list">
 +
              <ul>
 +
                <li>[1]&#160;&#160; DiCarlo J, Chavez A, Dietz S, Esvelt K, Church G. Safeguarding CRISPR-Cas9 gene drives in yeast. Nature Biotechnology [Internet]. 2015 [cited 10 October 2017];33(12):1250-1255. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4675690/</li>
 +
              </ul>
 +
            </div>
 +
          </div>
 +
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 +
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Latest revision as of 17:24, 30 October 2017

Chalmers Gothenburg iGEM 2017

Overview

The lung cancer biosensor was divided into two major parts: receptor integration and pathway. The native membrane localized GPCRs STE2 and STE3 were substituted with the two heterologous GPCRs Olf1258 and Ri7 in two different yeast mating types. These two heterologous GPCRs were fused with GFP to test membrane localization. Finally, the functionality of the GPCRs was tested using the plasmid pGFP, were PFUS1 is coupled to GFP. PFUS1 is a mating gene promoter induced after GPCR induction. For testing the functional pathway, the loxP-PTEF-loxP switch was investigated. Before the PTEF promoter is turned, GFP is expressed. Therefore, the decrease in GFP expression was tracked after the turning of the promoter, indicating that the promoter switch was successful.

Receptors

Integration of Olfr1258 and Ri7

For the biosensor to work, modifications of the two yeast strains, a and α, needed to be performed. One major modification was the substitution of the native GPCRs, STE2 and STE3, with the two heterogenous olfactory receptors, Ri7 and Olfr1258. This was executed using a CRISPR system with Cas9 and gRNA specific to STE2 and STE3. For more information see Project, constructs.

By using colony PCR, the substitution/integration of the GPCRs could be validated, after transformation of sequences for GPCR and CRISPR system. The colony PCRs showed successful results for the incorporation of both of the GPCRs into the genome of the respective yeast strain. Olfr1258 has been incorporated into the strain CENPK_111-61A and Ri7 into CENPK_11-11C.

Membrane localization

To enable visualization of the localization of the receptors in the yeast cells, the yeast was transformed with a plasmid containing the GPCRs fused together with GFP. The green fluorescence could then easily enable detection of the GPCRs. For the yeast strain with Olfr1258, the membrane localization of the GPCR could be verified. In the leftmost picture in Figure 1, it can be seen that Olfr1258 is clearly located in the outer membrane of the cell. In the rightmost picture, it can be seen that these cells fluoresce in the nuclear membrane which is clearly shown when the cells in this image is dividing. This image thereby suggest that the GPCR is localized in all membranes.

Figure 1.The yeast expressing Olfr1258 fused together with GFP. The image visualizes the membrane-localization of Olfr1258 due to the green fluorescence expressed at the edges of the yeast cells.

For the yeast cells expressing Ri7 fused together with GFP it could be visualized that this GPCR is membrane-localized as well, see Figure 2.

Figure 2.The yeast expressing Ri7 fused together with GFP. The image visualizes that Ri7 is membrane-localized due to the green fluorescence expressed at the edges of the yeast cell.

Lethality of VOCSs in unmodified strain

A lethality test of the VOCs in unmodified yeast strain was performed to be able to examine what range of concentrations the cells could handle and survive in. This test was crucial for the project since the concentration that could be used in the other test of the VOCs together with the modified strain needed to be in a concentration that was known not to affect the cell growth.

Figure 3. The lethality test for butanone showing the OD of the yeast cells over approximately 5 hours after exposure to different concentrations of butanone and DMSO.

This experiment was designed to expose the cells to different concentrations of respective VOC as well as different amounts of DMSO. The OD was measured after 2, 4 and 5,5 hours.

From the lethality test for butanone it could be seen that this range of concentrations, the butanone itself did not kill the cells. The cells that were exposed to 1 μM (1% DMSO), 10 μM (1% DMSO),1 mM (0.1% DMSO) and 10 mM (1% DMSO) did grow without being affected. However, the cells that were exposed to a higher concentration of DMSO had a clear decreased growth rate.

Figure 4. The lethality test for n-octanal showing the OD over approximately 5 hours after exposure to different concentrations of octanal and DMSO.

n-Octanal showed to have more negative impact on the lethality of the cells. The cells that were exposed to 10 μM (1% DMSO) and 1 μM (1% DMSO) had the highest survival. The cells that were exposed to 100 μM (10% DMSO), 10 mM (1% DMSO) and the DMSO control (10% DMSO) had a clear decreased growth rate. However, the 1 mM (0.1% DMSO) showed a weak growth and it was clearly visualized that these cells were affected.

From these two lethality test, it can be concluded that the concentration of DMSO is crucial for the survival of the cells and the concentration should be below 1%. The concentration range tested for butanone did not affect the cells. However, the concentration range tested for n-octanal had an effect on the cells and the concentration of octanal should be kept between 1 μM and 10 μM for the cells to stay unaffected.

BioLector

The GPCRs’ ability to recognize and bind the VOCs, initiate the MAPK pathway and thus activate the PFUS1 could be evaluated with a BioLector. Yeast with Ri7 and Olfr1258 incorporated was induced with n-octanal and butanone, respectively. A plasmid with GFP, pGFP, under the control of PFUS1 was incorporated and thus, GFP would be expressed as a positive output.

The result for Olfr1258 shows a slightly higher GFP expression when the yeast is induced with butanone, see Figure 5. For Ri7 there is no clear result. The higher GFP expression for Olfr1258 indicates that the receptor is able to sense the butanone and initiate the MAPK pathway which in turn activates the PFUS1 promoter. However, one need to consider the possibility that such differences might occur due to varying growth rates compared to the cells’ autofluorescence and further tests would be needed. If the further tests strengthen the results, it shows that the Olfr1258 works as it should.

Figure 5.The results from the BioLector of Olfr1258. The different results for respective concentrations are plotted with GFP emission per OD at the y-axis against time on the x-axis.

Pathway

Cre-Cas9

The Cre-Cas9 construct was successfully assembled together into a plasmid. To make the PFUS1 promoter activate, pheromones are needed since PFUS1 activation is accomplished through the yeast pheromone pathway. Pheromones are produced through the mating mechanism of the yeast. The activation of PFUS1 promoter will lead to expression of Cre-recombinase so the LoxP-sites would switch. The switch-system was designed so that it would be non-reversible and by flipping the promoter PTEF1, expression of the GFP would stop and Cas9 would be expressed instead.

The two different mating strains successfully mated which was done using selection and microscopic imaging. Further fluorescence microscopy images and colony-PCR runs were done to verify that the switch worked. The results came back negative. Sequencing of the Cre-Cas9 construct showed mutations in the Cre-recombinase part and might be the reason to why these experiments were unsuccessful.

To truly make sure that the flip worked and that the problem was not due to the deviation in the sequence of PCRE or that the PFUS1 promoter is too weak to induce expression, another test was done by using an inducible Cre-recombinase plasmid. The activation of Cre-recombinase in the plasmid is accomplished by the presence of galactose. The cell culture was grown in a Delft medium, containing galactose, and observed under a fluorescence microscope during three days. Three replicates were used and all gave a similar result. Figure 6 shows the fluorescence measurements during these three days for one colony. The GFP expression is clearly decreased with the passage of time.

Figure 6. A schematic view over the three days the cells were grown in galactose. The left figure show a microscopic image of the cells to give a sense of the amount of cells and the right figure show the fluorescence of these cells.

A colony-PCR was also performed for this test, in order to confirm that the flip worked at sequence level and to make sure that the decrease in expression comes from the switch and not something else. The gel electrophoresis, Figure 2, shows that the flip appear to have succeeded in one of the replicates tested. The clear band at 1300 bp indicates that the flip worked. The upper run shows a gel electrophoresis with only two primers. Only the cells who succeeded to perform the flip will show a band. The lower run shows a gel electrophoresis with three primers. If the flip did not succeed, a band at 500 bp will appear as opposed to 1300 bp, if the flip worked. Two colony-PCRs were run, due to the the fact that the flip might not have a 100 % efficiency, and since the PCR would favour the shorter construct. In the lower gel, there are two band shown for the second replicate, which indicate that the efficiency was not 100 %. Notable is that the other two replicates showed no bands at all in either test, which means that the PCR did not work for them. There should at least be a band in the lower run regardless of the direction of the promoter.

Figure 7.The gel electrophoresis result for the colony-PCR. In the first well the control is loaded, and the other three show the three replicates. Same goes for the lower run. A band at 1300 bp indicate a success in the flipping of the promoter, and a band at 500 bp show a failure. The ladder used is GeneRuler 1kb.

Cre-gRNA

No further tests was done on this pathway. Please check out Project, description or check out the reference for more information [1].

Future directions

For future directions of this project, there are some aspects to consider; firstly, to make all parts of the system work and get a complete functional system, from the sensing of the VOCs to the output of ADE2.

Once a functioning system is finalized, several steps are required to allow it to be taken into use. To get the biosensor approved for usage in a screening program, extensive testing of the method is required both on healthy people and lung cancer patients. This to be able to show that the system can distinguish between healthy and sick patients.

References

  • [1]   DiCarlo J, Chavez A, Dietz S, Esvelt K, Church G. Safeguarding CRISPR-Cas9 gene drives in yeast. Nature Biotechnology [Internet]. 2015 [cited 10 October 2017];33(12):1250-1255. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4675690/