Difference between revisions of "Template:Freiburg/CSS"

 
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Line 1: Line 1:
−
{{Freiburg/CSS}}
+
{{Freiburg/Javascript}}
−
{{Freiburg/Menubar}}
+
−
{{Freiburg/wiki_content_start}}
+
−
<!-- wiki content goes in here -->
+
  
 
<html>
 
<html>
  
 
<style>
 
<style>
−
/*========= BEGIN: style for navigation bar ==========*/
+
 
−
#results {
+
 
−
    background: url(https://static.igem.org/mediawiki/2015/2/2e/Freiburg_icon_results_active_yellow.png) no-repeat;
+
 
 +
/* =================== CSS Navigation =================== */
 +
/* Resetting css coming from wiki itself
 +
/* Clearing DokuWiki elements
 +
/* Declare Fonts for non-Windows machines
 +
/* CSS for Menubar
 +
/* Important Site setup
 +
/* Background
 +
/* Text Styling
 +
/* Full Width Banners
 +
/* Footer
 +
/* Accordion - Ausklappmenues
 +
/* Lightbox
 +
/* Float Barrier
 +
/*
 +
/* Flipcard-CSS for the members-section: inline in https://2015.igem.org/Team:Freiburg/Team
 +
/* CSS-slider: inline for the respective pages. e.g. https://2015.igem.org/wiki/index.php?title=Team:Freiburg/PaP/ScienceFair
 +
/* CSS classes and ids for Human Practice
 +
/* CSS for back-button
 +
/* CSS for the upwards-blob: on wiki_content_start
 +
/* CSS for Project Pages
 +
/* Horizontal Menu for Paragraphs
 +
/* Button for important links
 +
/* =================== END: CSS Navigation =================== */
 +
 
 +
 
 +
 
 +
/* ============= Resetting css coming from wiki itself ============= */
 +
body {
 +
    font: sans-serif;
 +
    color: #000;
 +
    margin: 0px;
 +
    padding: 0px;
 +
    background-color: #FCFCFC;
 +
line-height: 1.8;
 
}
 
}
  
−
#results a {
+
a {
−
     color: #ecdc18;
+
     color: #002BB8;
 +
    text-decoration: none;
 +
    background: transparent none repeat scroll 0% 0%;
 
}
 
}
  
−
#runningchip {
+
p {
−
     left: 52%;
+
     margin: 0.4em 0px 0.5em;
 +
    line-height: 1.5em;
 
}
 
}
−
/*========= END: style for navigation bar ==========*/
 
  
−
.image_box.left {
+
#globalWrapper {
−
     margin-right: 50px;
+
     position: absolute;
 +
    font-size: 150%;
 +
    width: 100%;
 +
    margin: 0px;
 +
    padding: 0px 0px 0px;
 
}
 
}
  
−
.image_box.right {
+
#mainContainer {
−
     margin-left: 50px;
+
     margin: 0;
 +
    width: 100%;
 
}
 
}
  
 +
#top_menu_under {
 +
    position: relative;
 +
    width: 100%;
 +
    height: 16px;
 +
    border-bottom: 2px solid #000;
 +
}
  
−
/* ========== Style for Details-Boxes ================*/
+
#top_menu_14 {
 +
    position: fixed;
 +
    width: 100%;
 +
    top: 0px;
 +
    left: 0px;
 +
    height: 16px;
 +
    background-color: #383838;
 +
    border-bottom: 2px solid #000;
 +
    z-index: 9999;
 +
}
  
−
.closeButton,
+
 
−
.details_box_trigger{
+
#top_menu_inside {
−
     display:inline-block;
+
    display: block;
−
     transition:all linear 0.15s;
+
    position: relative;
−
     text-decoration: none !important;
+
    width: 900px;
−
     color: #0051A2;
+
    height: 100%;
−
     font-weight: 500;
+
    margin: 0px auto;
−
     float: right;
+
    padding: 0px 10px;
−
     cursor: pointer;
+
    border-left: 1px solid #C8C8C8;
−
     cursor: hand;
+
    border-right: 1px solid #C8C8C8;
−
     font-size: 20px;
+
    font-family: "arial",sans-serif;
 +
    font-size: 12px;
 +
    font-weight: 400;
 +
    color: #FFF;
 +
}
 +
 
 +
 
 +
 
 +
#content {
 +
    position: relative;
 +
    width: 100%;
 +
    margin: 0;
 +
    padding: 0;
 +
    background: #FFF none repeat scroll 0% 0%;
 +
    color: #000;
 +
    border-left: 0;
 +
    border-right: 0;
 +
  /* line-height: 0; */
 +
    z-index: 2;
 +
 
 +
 
 +
}
 +
 
 +
.visualClear {
 +
     clear: both;
 +
}
 +
 
 +
 
 +
/* removes URL from current site from the top of the window */
 +
.firstHeading{
 +
    font-size: 0;
 +
     border: 0;
 +
    margin-bottom: -8px;
 +
    margin-top: 0;
 +
}
 +
 
 +
/* ============= END: Resetting css coming from wiki itself ============= */
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
/* ============= Clearing DokuWiki elements ============= */
 +
 
 +
.tags{
 +
/* remove tags from bottom */
 +
    font-size: 0;
 +
}
 +
 
 +
acronym{
 +
/* remove text-decoration from acronyms*/
 +
    border-bottom: none;
 +
     text-decoration: none;
 +
    cursor: auto;
 +
}
 +
 
 +
.urlextern {
 +
    background: none !important;
 +
}
 +
 
 +
.link-https {
 +
     background: none !important;
 +
}
 +
 
 +
 
 +
/* ============= END: Clearing DokuWiki elements ============= */
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
/* ============= Declaring Fonts  ============= */
 +
 
 +
@font-face {
 +
     font-family: Roboto;
 +
     src: url(https://static.igem.org/mediawiki/2015/e/ee/Freiburg_Roboto-Regular.eot);
 +
     src: url(https://static.igem.org/mediawiki/2015/b/bf/Freiburg_Roboto-Regular-webfont.woff) format('woff'),
 +
     url(https://static.igem.org/mediawiki/2015/c/c3/Freiburg_Roboto-Regular.ttf) format('truetype'),
 +
     url('https://static.igem.org/mediawiki/2015/3/38/Freiburg_Roboto-Regular-webfont.svg#RobotoRegular') format('svg');
 +
}
 +
@font-face {
 +
  font-family: Roboto;
 +
  src: url(https://static.igem.org/mediawiki/2015/7/7f/Freiburg_Roboto-Light.eot);
 +
  src: url(https://static.igem.org/mediawiki/2015/2/28/Freiburg_Roboto-Light-webfont.woff) format('woff'),
 +
  url(https://static.igem.org/mediawiki/2015/6/64/Freiburg_Roboto-Light.ttf)  format('truetype'),
 +
  url('https://static.igem.org/mediawiki/2015/8/87/Freiburg_Roboto-Light-webfont.svg#RobotoLight') format('svg');
 +
  font-style: normal;
 
   font-weight: 300;
 
   font-weight: 300;
 +
 
 
}
 
}
  
 +
@font-face {
 +
    font-family: Roboto;
 +
    src: url(https://static.igem.org/mediawiki/2015/c/c4/Freiburg_Roboto-LightItalic-webfont.eot);
 +
    src: url(https://static.igem.org/mediawiki/2015/4/4b/Freiburg_Roboto-LightItalic-webfont.woff) format('woff'),        url(https://static.igem.org/mediawiki/2015/0/0e/Freiburg_Roboto-LightItalic-webfont.ttf)  format('truetype'),
 +
    url('https://static.igem.org/mediawiki/2015/1/1d/Freiburg_Roboto-LightItalic-webfont.svg#RobotoItalic') format('svg');
 +
    font-weight: normal;
 +
    font-style: italic;
 +
}
  
−
.details_box_trigger.active {
+
@font-face {
 +
    font-family: Roboto;
 +
    src: url(https://static.igem.org/mediawiki/2015/4/40/Freiburg_Roboto-Bold.eot);
 +
    src: url(https://static.igem.org/mediawiki/2015/e/e4/Freiburg_Roboto-Bold-webfont.woff) format('woff'),
 +
    url(https://static.igem.org/mediawiki/2015/b/b9/Freiburg_Roboto-Bold.ttf)  format('truetype'),
 +
    url('https://static.igem.org/mediawiki/2015/4/40/Freiburg_Roboto-Bold-webfont.svg#RobotoBold') format('svg');
 +
    font-weight: bold;
 +
    font-style: normal;
 
}
 
}
  
−
.details_boxes{
+
@font-face {
−
     padding:15px;
+
     font-family: Roboto;
−
     display: none;
+
     src: url(https://static.igem.org/mediawiki/2015/c/ce/Freiburg_Roboto-BoldItalic.eot);
−
     overflow:hidden;
+
     src: url(https://static.igem.org/mediawiki/2015/8/89/Freiburg_Roboto-BoldItalic-webfont.woff)  format('woff'),
−
     box-shadow:0px 1px 3px rgba(0,0,0,0.25);
+
     url(https://static.igem.org/mediawiki/2015/2/2f/Freiburg_Roboto-BoldItalic.ttf)  format('truetype'),
−
     border-radius:20px;
+
    url('https://static.igem.org/mediawiki/2015/a/ac/Freiburg_Roboto-BoldItalic-webfont.svg#RobotoBoldItalic') format('svg');
−
     background:#EEE;
+
     font-weight: bold;
 +
     font-style: italic;
 
}
 
}
  
−
.hovertext{
+
 
−
    visibility: hidden;
+
 
−
    opacity: 0;
+
 
−
    position: absolute;
+
/* ============= END: Declaring Fonts  ============= */
−
    font-size: 120%;
+
 
−
    font-weight: 500;
+
 
−
    color: #0051A2;
+
 
−
    transition: opacity 0.5s ease-in;
+
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
/* ============= Important Site setup ============= */
 +
 
 +
 
 +
/* avoid dotted outline after clicking on links */
 +
a {
 +
  outline: 0;
 
}
 
}
  
−
#binding{
+
/* properties for central text box */
−
  left: 750px;
+
.bodyContent{
−
  top: 230px;
+
    overflow: hidden;
 +
    box-sizing: border-box;
 +
/* REMOVE WITH NEW NAVIGATION */
 +
/* Avoid layout damage due to old navigation */
 +
    clear: both;
 +
}
 +
/*adds padding on topside - necessary for attached menubar: mauri*/
 +
.content_background_wrapper{
 +
padding-top: 100px;
 
}
 
}
  
−
#binding:hover + #binding_label {
+
 
−
     transform: translateY(-40px);
+
/* flows around all the content in the center of the screen */
−
     opacity: 1;
+
.page_content{
−
    visibility: visible;
+
     margin: 20px 5px;
 +
     box-sizing: border-box; /* allows for efficient paositioning of the elements within */
 
}
 
}
  
−
#cellfree{
+
/* flows around text in the page_content */
−
  left: 360px;
+
.content_box{
−
  top: 150px;
+
    border-radius: 30px 60px;
 +
    overflow: hidden;
 +
    padding: 4%;
 +
    margin: 2em 10px;
 
}
 
}
 +
/*links linking to ID's have to link to a span located on top of the targeted id. This span has to be an anchor element, otherwise the menubar will float over the targeted headline*/
 +
.anchor{
 +
display: block;
 +
  height: 62px; /*same height as header*/
 +
  margin-top: -62px; /*same height as header*/
 +
  visibility: hidden;
 +
}
 +
 +
/* ================== FLEXBOXES and IMAGE_BOXES ===================*/
  
−
#cellfree:hover + #cellfree_label {
+
/* flexbox: contains the whole dokuwiki image part and renders it readable
−
     transform: translateY(-40px);
+
/* flexbox images are placed in one row and break the text.
−
     opacity: 1;
+
/* allow placing of several images including caption in one row */
−
     visibility: visible;
+
.flexbox .thumb2 .thumbinner .thumbcaption { /*circumvents the iGEM tag thumbcaption*/
 +
     font-size: 70%;
 +
     padding: 0 10px;
 +
     text-align: justify;
 
}
 
}
  
−
#immobilization{
+
.flexbox img,
−
  left: 90px;
+
.image_box img {
−
  top: 10px;
+
    max-width: 100%;
 
}
 
}
  
−
#immobilization:hover + #immobilization_label {
+
.image_box .thumb2,
−
     transform: translateY(-40px);
+
.image_box .thumb2 .thumbinner,
−
    opacity: 1;
+
.flexbox .thumb2,
−
    visibility: visible;
+
.flexbox .thumb2 .thumbinner{
 +
     max-width: 80vw;
 
}
 
}
  
−
#irif{
+
/*
−
  left: 80px;
+
.image_box .thumb2 .thumbinner img{
−
  top: 290px;
+
    width: 100%;
 +
}*/
 +
 
 +
/* see: https://css-tricks.com/snippets/css/a-guide-to-flexbox/*/
 +
 
 +
.flexbox{
 +
    display: -moz-box;
 +
    display: -ms-flexbox;
 +
    display: -webkit-box;
 +
    display: -webkit-flex;
 +
    display: flex;
 +
    margin: 5% 0;
 +
    -moz-justify-content: space-around;
 +
    -ms-justify-content: space-around;
 +
    -webkit-justify-content: space-around;
 +
    justify-content: space-around;
 +
    -moz-flex-wrap: wrap;
 +
    -ms-flex-wrap: wrap;
 +
    -webkit-flex-wrap: wrap;
 +
    flex-wrap: wrap;
 +
    align-content: center;
 
}
 
}
  
−
#irif:hover + #irif_label {
+
.flexbox .thumbinner{
−
    transform: translateY(-40px);
+
  margin: 0 5% 5% 5%;
−
    opacity: 1;
+
−
    visibility: visible;
+
 
}
 
}
  
−
#multbind{
+
/* for DokuWiki image boxes this prevents a zickzack positioning of the flexbox childs when wrap occurs */
−
   left: 430px;
+
a.media{
−
  top: 230px;
+
   margin: 0 auto;
 
}
 
}
  
−
#multbind:hover + #multbind_label {
+
/* contains images where text shall float around */
−
     transform: translateY(-40px);
+
.image_box .thumb2 .thumbinner .thumbcaption{
−
     opacity: 1;
+
     margin: 0.4em 0;
−
     visibility: visible;
+
     overflow: hidden;
 +
     font-size: 70%;
 +
    text-align: justify;
 
}
 
}
  
−
#salmonella{
+
.image_box.left{
−
  left: 750px;
+
    float: left;
−
  top: 230px;
+
    margin-right: 3%;
 
}
 
}
  
−
#salmonella:hover + #salmonella_label {
+
.image_box.right{
−
     transform: translateY(-40px);
+
     float: right;
−
     opacity: 1;
+
     margin-left: 3%
−
    visibility: visible;
+
 
}
 
}
  
−
.circle:hover{
+
@media screen and (max-width: 600px){
−
     transition: all linear 0.5s;
+
 
−
     transform: translate(-25px, -25px);
+
.image_box{
−
     width: 150px;
+
     display: -moz-box;
−
     height: 150px;
+
     display: -ms-flexbox;
−
     border-radius: 100px;
+
     display: flex;
 +
     display: -webkit-box;
 +
     -ms-justify-content: space-around;
 +
    -moz-justify-content: space-around;
 +
    -webkit-justify-content: space-around;
 +
    justify-content: space-around;
 +
    -moz-flex-wrap: wrap;
 +
    -ms-flex-wrap: wrap;
 +
    flex-wrap: wrap;
 +
    -webkit-flex-wrap: wrap;
 +
    align-content: center;
 +
    font-size: 70%;
 +
    padding: 0;
 +
    text-align: justify;
 +
    width: 100%;
 
}
 
}
  
−
/* ========== END: Style for Details-Boxes ================*/
+
} /* end media query */
  
 +
/* ================== END: FLEXBOXES and IMAGE_BOXES ===================*/
  
−
/* ========== Style for Circles in animated box ========== */
 
  
−
.circle{
+
/* prevents the floating of images out of the DokuWiki-sections */
−
    width: 100px;
+
.level1,
−
    height: 100px;
+
.level2{
−
    border-radius: 50px;
+
  overflow: hidden;
−
    border: 3px solid white;
+
−
    position: absolute;
+
 
}
 
}
  
−
.link_image{
+
 
−
     position: relative;
+
/* references coming from DokuWiki */
 +
.footnotes{
 +
     width: 90%;
 +
    margin: 2em auto 0 auto;
 
}
 
}
−
.results_headline{
+
 
−
color: #C5162F;
+
.fn_top{
−
font-size: 40px;
+
  font-size: 70%
−
text-align: right;
+
−
margin-bottom: 28px;
+
−
margin-top: 20px;
+
−
line-height: 45px;
+
 
}
 
}
−
.headline_left{
+
 
−
  text-align: left;
+
/* styling tables coming from dokuwiki */
 +
.content_box .table{
 +
    margin: 2em 5%;
 
}
 
}
−
.kommentar{
+
 
−
  display:none;
+
/* the element that can be used to style the table */
 +
.content_box .table .inline{
 +
    font-size: 90%;
 +
    width: 100%;
 +
    border-collapse: collapse;
 
}
 
}
−
#device_results, #agfp_results, #specific_surfaces,#own_mix{
+
 
−
  margin-top:100px;
+
/* grey background for the header line */
 +
.content_box .table th{
 +
    background-color: #DDD;
 
}
 
}
−
#anti_tetanus_results{
+
 
−
  margin-top:50px;
+
/* greyish background for every second row */
 +
.content_box .table tr:nth-child(odd){
 +
    background-color: #EEE;
 
}
 
}
  
−
/* ========== END: Style for Circles in animated box ========== */
+
/* move text away from cell borders */
 +
.content_box .table td,
 +
.content_box .table th{
 +
    padding: 0 10px;
 +
}
  
−
/* ========== Remove Explore the DiaCHIP on small devices =================*/
+
/* hovering efects */
 +
.content_box .table tr:hover{
 +
    background-color: #D2D4F3;
 +
}
  
−
@media screen and (max-width: 450px){
+
/* === content_box2 for brick layout === */
−
     #explore_DiaCHIP{
+
 
−
         display: none;
+
/* two content boxes in same line (sb) */
 +
.content_box2_parent {
 +
    text-align: center;
 +
}
 +
 
 +
.content_box2_child {               
 +
    border-radius: 30px 60px;
 +
    display: inline-block;
 +
    background-color: #FFFFFF;
 +
    text-align: justify;
 +
    vertical-align: middle;
 +
    box-shadow: 1px 1px 10px #888;
 +
    -webkit-box-shadow: 1px 1px 10px #888;
 +
    -moz-box-shadow: 1px 1px 10px #888;
 +
    padding: 4%;
 +
    min-height: 430px;
 +
}
 +
 
 +
@media screen and (min-width: 601px){
 +
     .content_box2_child{
 +
         margin: 2em 1em 2em 1em;
 +
        width: 35%;
 
     }
 
     }
 +
}
  
−
     #anti_tetanus_results, #device_results, #agfp_results, #specific_surfaces,#own_mix{
+
@media screen and (max-width: 600px){
−
      margin-top:20px;
+
     .content_box2_child{
 +
        margin: 1em 0 0;
 +
        width: auto;
 
     }
 
     }
 +
}
 +
 +
/* === Floatboxes for two column layout === */
 +
 +
.floatbox{
 +
    width: 48%; /* 100% - 2*48% = 4% padding*/
 +
}
  
−
    .results_headline,
+
@media screen and (min-width: 601px){
−
     .headline_left{
+
     .floatbox.left{
−
      font-size: 120%;
+
        float: left;
−
      text-align: right;
+
−
      margin-bottom: 10px;
+
−
      margin-top: 20px;
+
−
      line-height: inherit;
+
 
     }
 
     }
  
 +
    .floatbox.right{
 +
        float: right;
 +
    }
 
}
 
}
  
 +
@media screen and (max-width: 600px){
 +
    .floatbox{
 +
        float: none;
 +
        width: auto;
 +
    }
 +
}
  
−
</style>
+
/* ============= END: Important Site setup ============= */
  
−
<script type="text/javascript">
 
  
−
// JQUERY for Details-Boxes
 
  
−
$(document).ready(function() {
+
 
−
     function close_accordion_section() {
+
 
−
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mehr Formulierungen wie: we demonstrated..., we (successfully) tested, we achieved...
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<div class="results_headline">Essential Results</div><p>Our aim was to develop a novel diagnostic tool enabling fast, simultaneous and label-free detection of antibodies in human blood sera. We successfully generated our self-established <a href="#own_mix">cell-free expression system</a> based on an <i>E. coli</i> lysate, which revealed an expression efficiency comparable to a commercial kit. To immobilize the expressed proteins, we established a <a href="#specific_surfaces">surface</a> specifically binding our target proteins.
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This system was used to produce a protein array that can be flushed with a serum sample. The binding of antibodies is detected in a label-free manner by an interferometric method called iRIf (imaging reflectometric interference) in real-time. Moreover, we <a href="#device_results">rebuilt the detection device</a> in a simplified and cost-efficient manner and thus, made it available for future iGEM Teams.
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Using the DiaCHIP, we did not only show the presence of antibodies in distinct solutions but also verified them in complex samples as human blood serum.
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            Video 1: Detection of anti-tetanus antibodies in human serum.
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  <div class="results_headline headline_left">Detection of Anti-Tetanus Antibodies in Human Blood Serum</div>
 
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    To test the DiaCHIP under real-life conditions, we analyzed the blood serum of a vaccinated person for the presence of anti-tetanus antibodies. We compared blood samples from before and after vaccination and could directly detect its effect. To capture the antibodies, the <a href="http://parts.igem.org/Part:BBa_K1621003">corresponding antigen</a> was expressed in <i>E. coli</i>, purified by His-tag affinity purification and spotted on a specific Ni-NTA surface. By comparing the spotted pattern with the localization of signal as detected by iRIf, the system's specificity even for complex samples was shown.
 
  
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            <strong>Figure 1: Western Blot analysis of purified tetanus antigen.</strong>
 
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            <strong>Figure 2: Quotient picture of iRIf measurement of human serum sample taken before vaccination.</strong>
 
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        The <a href=”http://parts.igem.org/Part:BBa_K1621003”>antigen</a> used for detection of anti-tetanus antibodies is derived from the tetanus neurotoxin and commonly used for <i>in vitro</i> testing. For preparation of the DiaCHIP, this antigen had to be overexpressed in <i>Escherichia coli</i> and purified from the whole cell lysate. To verify successful purification of the antigen Western Blot analysis was performed (figure 1). The usage of an antibody targeting the N-terminal His-tag revealed a protein with a molecular weight of about 50 kDa. This correlates with the expected molecular weight of the antigen <sup><a class="fn_top" href="#fn__1" id="fnt__1" name="fnt__1">1)</a></sup>. </br>
 
  
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Before we performed an iRIf measurement with the blood of the freshly vaccinated person we checked if we could detect an interaction of antibodies present in the blood with our purified tetanus antigen by an ELISA.
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Therefore, we spotted tetanus antigen and bBSA of the same concentration (12.5&nbsp;µg/mL) and twice PBS as negative controls. The tetanus antigen and one PBS well were incubated with 5&nbsp;mg/mL BSA, blood serum and HRP-labeled anti-human for 1&nbsp;h respectively. The bBSA and the other PBS well were incubated with HRP-labeled Streptavidine (0.5&nbsp;µg/mL) for 1 &nbsp;h (positive control). After each step the wells were washed with PBS. To visualize the binding, Tetramethylbenzidine was added and the absorption at 650&nbsp;nm over time was measured in a plate reader. The graph in figure 3 shows that the signal for tetanus is even higher than the positive control, which illustrates the successful binding of antibodies from the blood to tetanus antigens.
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                <strong>Figure 3: ELISA for the tetanus antigen with freshly vaccinated blood. The graph shows the absorption over time for TMB. The red line shows the binding of anti-tetanus antibodies out of the blood serum to purified tetanus antigen and the blue line shows the binding of HRP-Streptavidine to bBSA. The grey lines show the corresponding negative controls.</strong>
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        The purified antigen was immobilized on a Ni-NTA surface that specifically binds His-tagged proteins. Look<a href=”https://2015.igem.org/Team:Freiburg/Results/Binding”> here</a> to read more about the establishment of this surface. As a positive control, self-purified His-tagged GFP was immobilized at another spot. The reliable detection of anti-GFP was shown before (link). No binding event should be detected at the negative control spot that was covered with <strong>bBSA/mCherry (??)</strong>.
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                <strong>Figure 4: Quotient picture of iRIf measurement of human serum sample taken <b>three????</b> weeks after vaccination.</strong>
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                <strong>PLATZHALTER! Figure 5: Binding curve of the tetanus measurements.</strong>
 
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        The human serum samples we obtained have been taken before and <strong>three?</strong> weeks after a patient received a vaccination boost against tetanus. Analysis of the samples was performed using the same array set-up. Figures 2 and 4 show quotient pictures representing changes in the thickness of the layer covering the slide while it was flushed with the respective samples. The negative control spots do not indicate any binding events, while the binding of anti-GFP to the positive control spot verifies that both experiments are reliable. The comparison of the antigen spots shows that we specifically detected anti-tetanus antibodies in human serum in response to vaccination. No binding event was detected by flushing the array with the sample assumed to be negative (figure 2), whereas a distinct spot is visible after flushing it with the positive sample (figure 4). Both measurements in real-time are shown in parallel in video 1.</br>
 
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        The increase in relative light intensity at distinct spots over the course of the experiment is visualized in figure 5 and can be correlated with the amount of antibodies binding to the respective antigen spot. This indicates that the DiaCHIP enables quantification of antibody titers in addition to detecting their presence.
 
  
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        <br>
 
−
        Besides tetanus, other antigens of immunological relevance were taken into account. See all the results we obtained in terms of <a href="https://2015.igem.org/Team:Freiburg/Results/Diagnostics">diagnostics</a>.
 
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<div class="fn"><sup><a class="fn_bot" href="#fnt__1" id="fn__1" name="fn__1">1)</a></sup>
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<a class="urlextern" href="http://ac.els-cdn.com/S0171298510001567/1-s2.0-S0171298510001567-main.pdf?_tid=05754f9e-4f34-11e5-95a1-00000aacb360&amp;acdnat=1440952235_9b35c0a0389b3dbe95d48a4e8a583234" rel="nofollow" target="_Blank" title="http://ac.els-cdn.com/S0171298510001567/1-s2.0-S0171298510001567-main.pdf?_tid=05754f9e-4f34-11e5-95a1-00000aacb360&amp;acdnat=1440952235_9b35c0a0389b3dbe95d48a4e8a583234">Rui et al., 2011. Enhanced expression of soluble recombinant tetanus neurotoxin Hc in Escherichia coli as a tetanus vaccine candidate. Immunobiology.</a></div>
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    <div class="kommentar"> hier noch rein bringen, dass ihr euren eigenen cell-free mix hergestellt habt und mit diesem auch GFP expremieren könnt und es im array erfolgreich nachweisen könnt
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Auch, dass euer Mix low-cost ist, und trotzdem auf dem Niveau (oder besser) eines kommerziellen kits liegt - Community gedanke etc.  (ps12092015)</div>
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      <div class="thumbcaption">Video 2: Binding of anti-GFP antibodies to GFP-His on a Ni-NTA surface. Anti-GFP antibodies bind both to purified GFP-His and cell-free expressed GFP-His. Control spots consisting of cell-free expression mix without DNA template show no binding.</div>
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        Detection of Anti-GFP Antibodies Using Cell-Free Expressed GFP on Ni-NTA
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    Our self-made cell-free expression, the DiaMIX, in combination with specific glass surfaces allows the expression and purification of target proteins in a simple manner. The mix, after expressing His-tagged GFP, was spotted on a Ni-NTA coated glass slide. Even after several washing steps it could be shown that the proteins maintain their antibody binding properties. A GFP antibody solution was flushed over the slide and a specific binding could be detected in iRIf.
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      We could successfully show that the cell-free expression of GFP-His results in functional GFP proteins that are bound by anti-GFP antibodies during an iRIf measurent.
 
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The DNA coding for GFP fused to a 10x His-tag was added to the <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/Cellfree_Expression#diamix" title="DiaMIX">DiaMIX</a> and expression was performed for 2 h. The DiaMIX containing cell-free expressed GFP-His was then spotted on an iRIf slide with a Ni-NTA surface by hand (figure 1).
 
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This specific surface allows binding of the expressed GFP-His via the His-tag while all the other proteins present in the DiaMIX are not bound to the surface.
 
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The iRIf slide was then flushed with a anti-GFP antibody solution, to analyze the binding to the cell-free expressed GFP-His. As a positive control we spotted a purified GFP-His onto the surface as well as DiaMIX that did not contain any DNA as negative control (figure 2).
 
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            <strong>Figure 2: The two spots on the right are the cell-free expressed GFP-His proteins spotted onto the Ni-NTA surface. The spot on the left shows the positive control, GFP-His expressed in <i>E. coli</i>, purified and spotted onto the Ni-NTA surface.</strong>
 
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        The measurement shows a high signal for the cell-free expressed GFP-His and the positive control as can also be seen in the respective binding curve shwown in figure 3.
 
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        <div class="thumb2 trien" style="width:410px"><div class="thumbinner"><a class="lightbox_trigger" href="https://static.igem.org/mediawiki/2015/6/6d/Freiburg_2015_freiburg_cellfex_gfp_on_ni-nta_binding_curve.png"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/6/6d/Freiburg_2015_freiburg_cellfex_gfp_on_ni-nta_binding_curve.png" width="400"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=results_overview&amp;media=2015_freiburg_cellfex_gfp_on_ni-nta_binding_curve.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div><strong>Figure 3: Binding curve of anti-GFP binding to cell-free expressed GFP-His on a Ni-NTA surface.</strong> Cell-free expressed GFP-His was spotted onto a specific Ni-NTA surface and flushed with anti-GFP. The binding curve indicated a binding event at the respective spot.</div></div></div>
 
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Find out more about the <a href="https://2015.igem.org/Team:Freiburg/Results#assembly_pic">preparation</a> of the DiaCHIP by producing a protein microarray from a DNA template using cell-free expression.
 
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                  Figure 2: Functional and low-cost variant of the measuring device.
 
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        Building Our Low-Cost DiaCHIP Measuring Device
 
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        Commercial systems using the iRIf technology (imaging reflectomretric interference) are mostly bulky and expensive machines even though the components they are based on are rather simple. So we decided on building our own device consisting of not much more than two lenses, a LED and a camera. With this system we were able to reliably detect the binding of anti-GFP to GFP, thus confirming a detection sensitivity in the range of protein-protein interactions. To enable future iGEM Teams to profit from this device as we did, we provide all plans and parts necessary to rebuild it on this website for everyonbe to download and use.
 
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            <div class="thumbcaption">Video 1: Demonstration of the capability of our iRIf device at measuring antibody-antigen interaction. GFP was spotted on the left side, rabbit proteins (anti-HCV antibodies from rabbit) on the right.
 
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            To test our device we aimed at reproducing measurements that we were previously able to perform in our commercial device. For this reason we focused on using two antibodies: anti-GFP and polyclonal anti-rabbit. GFP and rabbit derived anti-HCV (Hepatitis C Virus) antibodies were used as antigens in this experiment. Note that the anti-HCV antibodies only served as binding partners for the anti-rabbit antibodies, since no HCV proteins were used in this experiment.
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            The antigens were spotted onto an iRIf slide whose binding layer consisted of an APTES/PDITC surface. The spots on the slide were produced by pipetting 3.5 µl [1 mg/ml] rabbit-anti-HCV protein and 3.5 µl [1 mg/ml] GFP onto the slide and incubated overnight. After incubation the slide was blocked for 30 min in BSA solution.
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            A Canon 50D camera was used to record the measurement and was set to take one picture every 5 seconds. The exposure time was set in order for the pixels in the image to be approx. 80% of maximum light saturation before the solution was flushed onto the chip.
 
  
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            The antibody solutions were pipetted into the flow-chamber without the use of any microfluidic device. Instead, a syringe was loaded with 660 µl [5 µg/ml] anti-GFP antibody solution (diluted in PBS) and connected to the input pipe of our device. The content of the syringe was then slowly released via this pipe into the chamber of the device by gently dispensing the solution from the syringe. When the whole volume ran over the chip, the process was repeated with 660 µl [5 µg/ml] anti-rabbit antibody solution in the same way. The injection of both solutions was performed during approximately 45 minutes.
 
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                <strong>Figure 3: Quotient picture of the same measurement with favourable light conditions.</strong>
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            Video 1 shows the results of the measurement. Both, binding of anti-GFP and anti-rabbit to the corresponding antigen spots, could be observed. Due to the fact that the experiment was performed on our demonstration device which was built with a transparent casing, fluctuations in surrounding light had a strong, detrimental influence on the measurement quality. To minimize the influence of the unstable surrounding light, the resulting pictures had to be averaged over 10 pictures each. This in turn lead to a more stable light situation, however the signal strengh dropped as a consequence. Figure XY shows a quotient picture of the measurement where the light situation was temporarily appropriate. The problem of surrounding light scattering into the device can of course be overcome using a non-transparent casing.      
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                  <p>Figure 1: Cell-free expressed GFP-His spotted on a Ni-NTA slide. Cell-free expression was performed for 2 hours at 37°C. The positive control was purified GFP-His, the negative control was a sample of cell-free expression mix not containing DNA. </p>
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        Establishing a Highly Specific Ni-NTA Surface
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        Since there is a variety of proteins present in the cell-free mix a specific surface on the future protein array is essential. That is why we established our Ni-NTA surface for the immobilization of the antigens on the chip. All expression constructs contain a His-tag fused to the coding sequence, resulting in antigens that can bind specifically to our Ni-NTA surface. Compared to an unspecific surface (PDITC) we could show that this Ni-NTA surface allows efficient binding of the target protein and prevents unspecific binding of other proteins that are present in the cell-free mix.
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                        <p>Figure 2: Cell-free expressed GFP-His spotted on a PDITC slide. Cell-free expression was performed for 2 hours at 37°C. The positive control was purified GFP-His, the negative control was a sample of cell-free expression mix not containing DNA. </p>
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                For cell-free protein expression lots of different proteins, like RNA-polymerases, ribosomes and other <i>E. coli</i> proteins, are essential. So during the process of cell-free expression all these proteins are, besides the target protein, also present in the microfluidic chamber. To have a sufficient amount of target protein immobilized on the chip we established a specific surface chemistry on the glass slide. After testing several <a href="https://2015.igem.org/Team:Freiburg/Results/Surface">tag systems</a> we established a Ni-NTA surface because it worked best for us. Using a Ni-NTA covered glass slide (figure 1) we could increase the amount of bound cell-free expressed GFP-His compared to an unspecific surface (figure 2). In both experiments ____2.5 µg?!?____ of self purified GFP-His was used as positive control. The cell-free expression mix without DNA is supposed to result in no target protein and served as negative control. The cell-free reactions were performed for 2 hours at 37°C. The samples were pipetted by hand onto the surfaces and incubated on the slide overnight. In the iRIf device, the optical detection method we used, the slides were blocked with BSA and then flushed with anti-GFP antibodies. An increase in light intensity on the slide where the spotted protein was located represents a binding event. Due to less unspecific binding of proteins to the surface, the interaction of anti-GFP antibodies with cell-free expressed GFP-His is higher on the Ni-NTA surface.
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        Producing Our Cell-free Expression Mix
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        The mechanism we are using to copy a DNA array template into a protein array on demand is based on cell-free expression. During our project we successfully established our cell-free expression system, the DiaMIX, from scratch, starting with an <i>E. coli</i> lysate. With this mix we succeeded in expressing correctly folded GFP and luciferase. Comparison of the DiaMIX with a commercial cell-free expression kit revealed the great potential of our system!
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            Our DiaMIX was prepared based on a protocol of <div class="todo_box">??? (Ref.)</div>.
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It is a very complex system containing many different enzymes and chemicals for special purposes. An overview of cell-free expression systems and its components can be found on the<a class=”wikilink1” href=https://2015.igem.org/Team:Freiburg/Project/Cellfree_Expression>methodology page</a>. <br/>
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To investigate the efficiency of our self-prepared expression system we performed an experiment comparing it to a commercially available kit. In order to obtain reliable results we used the exact same vector, containing a sequence encoding GFP. Like this we were able to trace the amount of expressed GFP using a plate reader over a period of two hours. As a negative control, both mixes were treated equally in a second sample but did not contain any DNA. <br/>
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The evaluation is shown in figure 1 and is based on triplicates. <b>The data was normalized to the mean value of the measurement of air. The uncertainty was calculated using the standard deviation. ÜBERARBEITEN JE NACH GRAPH</b> <br/>
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picture; Figure 1: <b>Cell-free expression of GFP using the DiaMIX and a commercial kit.</b> The reaction was performed in a volume of 50 µl each and monitored every minute at 37°C over a period of two hours. Excitation at 480 nm, emission at 520 nm. <br/>
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Both expression systems were shown to successfully express the applied vector. There is a clear enhancement of light emission observable at 520nm for both mixes in comparison to the respective negative control. The expression of GFP using the DiaMIX seems to be slightly better than by using the commercial kit. Still, one has to mention the actual purpose of the commercial kit to express linear templates. However, as the basis protocol for our mix was optimized for circular templates, we used a circular vector for the experiment. <br/>
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Furthermore, as already shown above, we successfully verified the expression of GFP with our cell-free mix in an <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Results#agfp_results" title="agfp_results">iRIf measurement</a>. <br/>
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To obtain these final results, a lot of optimization and testing had to be done. If you want to know more about how we established the DiaMIX for the DiaCHIP, you can find detailed information about the most important experiments on the <a class=”wikilink1” href="https://2015.igem.org/Team:Freiburg/Results/Cellfree">cell-free expression results page</a>. <br/>
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<h1>Explore the DiaCHIP</h1>
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<p>For establishing our device, we optimized all steps from the immobilization of DNA on the silicone of the flow-chamber to the specific binding of the target proteins on the glass slide. This way, we generated protein arrays we could use to detect different antibodies in human and rabbit serum with the iRIf detection method. <br> Click on the images below to explore our experiments from expression to detection!</p>
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    text-align: center;
−
        <img src="https://static.igem.org/mediawiki/2015/5/55/Freiburg_150912_Resultsoverviewgrafik_Tet_Sal_GFP_RJ.png" width="100%">
+
    background-color: #0051A2;
−
     </div>
+
    border-radius: 30px 15px 15px 30px;
−
</div>
+
    border: 1px groove #004081;
−
<p> Click on one of the images to get a further insight on how we build our DiaCHIP </p>
+
    float: left;
 +
}
  
−
</div>
+
.white_link {
 +
    color: #FFF;
 +
}
 +
/* ============== END:CSS for back-button =============*/
  
−
</html>
+
</style>
  
−
<!-- wiki content ends here -->
+
<!-- INITIALIZE UPWARDS BLOB, SO ITS TOP REFERENCES TO DOCUMENT TOP -->
−
{{Freiburg/wiki_content_end}}
+
 
 +
<style>
 +
 
 +
/* upwards navigation blob */
 +
 
 +
#upwards_blob{
 +
    display: none;
 +
}
 +
 
 +
 
 +
/* min-width is chosen so the blob does just not interfere with the content box */
 +
/* 1021 content_box-width + 46 blob-width + 20 scrollbar + some buffer space */
 +
@media screen and (min-width: 1144px){
 +
    #upwards_blob{
 +
        transition: transform 0.5s ease;
 +
        display: inline;
 +
        position: absolute;
 +
        top: 900px;
 +
        right: 30px;
 +
        z-index: 1000;
 +
        transform: translateY(130px);
 +
    }
 +
}
 +
 
 +
</style>
 +
 
 +
<div id="upwards_blob"><a href="#globalWrapper"><img src="https://static.igem.org/mediawiki/2015/9/93/Freiburg_upwards_blob.png"></a></div>
 +
 
 +
<style>
 +
/* ============= BEGIN: CSS for Project Pages ================ */
 +
.content_text {
 +
    border-radius: 30px 60px;
 +
    overflow: hidden;
 +
    padding: 4%;
 +
    margin: 2em 0;
 +
    margin-bottom: 0;
 +
    text-align: justify;
 +
    color: #000;
 +
    width: 50%;
 +
}
 +
 
 +
.rightshift {
 +
    margin-right: -5%;
 +
    margin-left: 8%;
 +
}
 +
 
 +
.leftshift {
 +
    margin-left: -5%;
 +
    margin-right: 8%;
 +
}
 +
 
 +
.reference_box {
 +
    overflow: hidden;
 +
    border: 1px solid rgb(0, 0, 0);
 +
    border-radius: 10px;
 +
    background-color: #EEE;
 +
    text-align: justify;
 +
    box-shadow: 1px 1px 10px #888;
 +
    padding: 1% 2% 3%;
 +
    margin-top: 2em;
 +
    margin-right: auto;
 +
    margin-left: auto;
 +
    width: 80%;
 +
}
 +
 
 +
.h1_fancy {
 +
    text-align: center;
 +
    color: #888;
 +
    font-size: 300%;
 +
    text-shadow: 
 +
        1px 0px 1px #ccc, 0px 1px 1px #eee,
 +
        2px 1px 1px #ccc, 1px 2px 1px #eee,
 +
        3px 2px 1px #ccc, 2px 3px 1px #eee,
 +
        4px 3px 1px #ccc, 3px 4px 1px #eee,
 +
        5px 4px 1px #ccc, 4px 5px 1px #eee,
 +
        6px 5px 1px #ccc, 5px 6px 1px #eee,
 +
        7px 6px 1px #ccc;
 +
}
 +
/* ============= END: CSS for Project Pages ================ */
 +
 
 +
 
 +
/* ============= BEGIN: Horizontal Menu for Paragraphs ================ */
 +
.horizontal_menu {
 +
    margin: 0px auto;
 +
}
 +
 
 +
.horizontal_menu ul{
 +
    margin: 0px auto;
 +
    padding: 0px;
 +
    list-style: none;
 +
    text-align: center;
 +
}
 +
 
 +
.horizontal_menu li {
 +
    margin: 0px 5px auto;
 +
    padding: 1%;
 +
    display: inline;
 +
    text-align: center;
 +
    border: 2px groove #0051A2;
 +
    border-radius: 10px;
 +
    background-color: #0051A2;
 +
}
 +
 
 +
.horizontal_menu a {
 +
    color: #FFF;
 +
}
 +
/* ============= END: Horizontal Menu for Paragraphs ================ */
 +
 
 +
/* ============= Added: Styling for results pages ================ */
 +
 
 +
 
 +
.content_box h1{
 +
color: #C5162F;
 +
font-size: 40px;
 +
margin-bottom: 28px;
 +
margin-top: 20px;
 +
line-height: 45px;
 +
font-weight: 200;
 +
padding-top: 0;
 +
padding-bottom: 0;
 +
overflow: visible;
 +
text-align: center;
 +
}
 +
 
 +
.results_page h1{
 +
text-align: right;
 +
}
 +
 
 +
.content_box h2 {
 +
    font-size: 140%;
 +
    font-weight: 200;
 +
    letter-spacing: 0.5px;
 +
}
 +
 
 +
.results_page h2{
 +
letter-spacing: 0px;
 +
font-size: 35px;
 +
text-align: right;
 +
margin-bottom: 28px;
 +
margin-top: 20px;
 +
line-height: 45px;
 +
font-weight: 200;
 +
}
 +
 
 +
.content_box h3{
 +
font-weight: 200;
 +
 
 +
}
 +
 
 +
h2.left,
 +
h1.left{
 +
text-align: left;
 +
}
 +
 
 +
@media screen and (max-width: 600px){
 +
 
 +
.results_page h1{
 +
font-size: 120%;
 +
margin-bottom: 10px;
 +
margin-top: 8px;
 +
line-height: 150%;
 +
}
 +
 
 +
.results_page h2{
 +
font-size: 100%;
 +
margin-bottom: 10px;
 +
margin-top: 8px;
 +
line-height: 110%;
 +
}
 +
 
 +
/*========================= Style for Important Links Button =======================*/
 +
.link_button_arrow{
 +
background-image: url("https://static.igem.org/mediawiki/2015/f/ff/Freiburg_submenu_arrow.png");
 +
background-repeat: no-repeat;
 +
background-position: 3% 50%;
 +
background-size: 10px 13px;
 +
}
 +
 
 +
.link_button {
 +
margin: 0px auto;
 +
padding: 0px 5px 0px 10px;
 +
width: 200px;
 +
line-height: 16px;
 +
text-align: center;
 +
background-color: #888;
 +
font-size: 14pt;
 +
border-radius: 2px;
 +
color: #FFF;
 +
opacity: 0.8;
 +
box-shadow: 1px 1px 1px 2px #777;
 +
}
 +
 
 +
.link_button a:hover {
 +
    text-decoration: underline;
 +
}
 +
 
 +
.link_button a {
 +
    color: #FFF;
 +
}
 +
</style>
 +
 
 +
 
 +
</html>

Latest revision as of 01:45, 19 September 2015