Difference between revisions of "Team:Freiburg/Testpage"

Line 1: Line 1:
 
{{Freiburg/CSS}}
 
{{Freiburg/CSS}}
 
{{Freiburg/Menubar}}
 
{{Freiburg/Menubar}}
 +
 
{{Freiburg/wiki_content_start}}
 
{{Freiburg/wiki_content_start}}
−
<!-- wiki content goes in here -->
 
−
 
 
<html>
 
<html>
  
 
<style>
 
<style>
−
/*========= BEGIN: style for navigation bar ==========*/
+
/*======= BEGIN: General Styling ========*/
−
#results {
+
body {
−
     background: url(https://static.igem.org/mediawiki/2015/2/2e/Freiburg_icon_results_active_yellow.png) no-repeat;
+
     color: #000;
 
}
 
}
  
−
#results a {
+
.page_content {
−
     color: #ecdc18;
+
     margin-bottom: 0px;
 
}
 
}
  
−
#runningchip {
+
.content_box3 {
−
     left: 52%;
+
     /*box-shadow: none;*/
 +
    overflow: hidden;
 +
    background-color: #FFF;
 +
    padding: 1% 2% 0% 2%;
 +
    border-radius: 30px 60px;
 +
    margin-top: 2em;
 
}
 
}
−
/*========= END: style for navigation bar ==========*/
 
  
−
.image_box.left {
+
h1 {
−
     margin-right: 50px;
+
     text-align: center;
 
}
 
}
  
−
.image_box.right {
+
.header_box {
−
     margin-left: 50px;
+
  display: none;
 +
    overflow: hidden;
 +
    padding: 2% 0%;
 +
     margin: auto;
 +
    width: 70%;
 +
    text-align: left;
 +
    /*background-color: #FFF;
 +
    border-radius: 30px 60px;
 +
    box-shadow: 1px 1px 10px #888;
 +
    -webkit-box-shadow: 1px 1px 10px #888;
 +
    -moz-box-shadow: 1px 1px 10px #888;*/
 
}
 
}
  
 +
.image_box {
 +
    margin-bottom 10%;
 +
    margin-left: -8%;
 +
    margin-top: -10%;
 +
    margin-right: -10%;
 +
}
 +
/*======= END: General Styling ========*/
  
−
/* ========== Style for Details-Boxes ================*/
 
  
−
.closeButton,
+
/*========= BEGIN: style for navigation bar ==========*/
−
.details_box_trigger{
+
#project {
−
     display:inline-block;
+
     background: url(https://static.igem.org/mediawiki/2015/d/da/Freiburg_icon_project_active_yellow.png) no-repeat;
−
    -webkit-transition:all linear 0.15s;
+
−
    -ms-transition:all linear 0.15s;
+
−
    transition:all linear 0.15s;
+
−
    text-decoration: none !important;
+
−
    color: #0051A2;
+
−
    font-weight: 500;
+
−
    float: right;
+
−
    cursor: pointer;
+
−
    cursor: hand;
+
−
    font-size: 110%;
+
−
    font-weight: 300;
+
 
}
 
}
  
−
 
+
#project a {
−
.details_box_trigger.active {
+
    color: #ecdc18;
 
}
 
}
  
−
.details_boxes{
+
#runningchip {
−
     padding:15px;
+
     left: 18.6%;
−
    display: none;
+
−
    overflow:hidden;
+
−
    box-shadow:0px 1px 3px rgba(0,0,0,0.25);
+
−
    border-radius:20px;
+
−
    background:#EEE;
+
 
}
 
}
 +
/*========= END: style for navigation bar ==========*/
  
−
.hovertext{
+
 
−
    visibility: hidden;
+
/*========= BEGIN: Linkbutton in slider ============*/
−
    opacity: 0;
+
.intro_button a:hover {
−
     position: absolute;
+
     text-decoration: underline;
−
    font-size: 120%;
+
−
    font-weight: 500;
+
−
    color: #0051A2;
+
−
    -ms-transition: opacity 0.5s ease-in;
+
−
    -webkit-transition: opacity 0.5s ease-in;
+
−
    transition: opacity 0.5s ease-in;
+
 
}
 
}
  
−
#binding{
+
.intro_button a {  
−
  left: 750px;
+
    color: #FFF;
−
  top: 230px;
+
 
}
 
}
  
−
#binding:hover + #binding_label {
+
.intro_button {
−
     transform: translateY(-40px);
+
     margin: 0px auto;
−
     -webkit-transform: translateY(-40px);
+
    padding: 2px 5px 5px 10px;
−
     -ms-transform: translateY(-40px);
+
    width: 180px;
−
     opacity: 1;
+
    line-height: 16px;
−
     visibility: visible;
+
     text-align: center;
 +
    /*background-color: #326aab;*/
 +
    background-color: #0051A2;
 +
     font-size: 14pt;
 +
    border-radius: 8px;
 +
    /*box-shadow: 1px -1px 1px 0px #888;*/
 +
     /*border: 1px solid #F9F9F9;*/
 +
     color: #FFF;
 
}
 
}
  
−
#cellfree{
+
.menu-arrow{
−
  left: 360px;
+
background-image: url("https://static.igem.org/mediawiki/2015/f/ff/Freiburg_submenu_arrow.png");
−
  top: 150px;
+
background-repeat: no-repeat;
 +
background-position: 5% 50%;
 +
background-size: 10px 13px;
 
}
 
}
 +
/*========= END: Linkbutton in slider ============*/
 +
 +
 +
/* code adapted from http://www.smashingmagazine.com/2012/04/pure-css3-cycling-slideshow/*/
  
−
#cellfree:hover + #cellfree_label {
+
#slider {
−
    transform: translateY(-40px);
+
  height: 500px;
−
    -webkit-transform: translateY(-40px);
+
  width: 910px;
−
    -ms-transform: translateY(-40px);
+
  overflow: visible;
−
    opacity: 1;
+
  position: relative;
−
    visibility: visible;
+
 
}
 
}
  
−
#immobilization{
+
/* hides everything outside the slider box */
−
  left: 90px;
+
#mask {
−
  top: 10px;
+
  overflow: hidden;
 +
  height: 500px;
 
}
 
}
  
−
#immobilization:hover + #immobilization_label {
+
/* initialize position for positioning slides outside the slider */
−
    transform: translateY(-40px);
+
#slider ul {
−
    -webkit-transform: translateY(-40px);
+
  margin: 0;
−
    -ms-transform: translateY(-40px);
+
  padding: 0;
−
    opacity: 1;
+
  position: relative;
−
    visibility: visible;
+
 
}
 
}
  
−
#irif{
+
/* define image properties and position them outside the slider mask */
−
  left: 80px;
+
#slider li {
−
  top: 290px;
+
  width: 900px;  /* Width slide */
 +
  height: 500px; /* Height slide */
 +
  position: absolute;
 +
  right: 0; /* Original Position - Outside of the Slider */
 +
  list-style: none;
 
}
 
}
  
−
#irif:hover + #irif_label {
+
 
−
    transform: translateY(-40px);
+
#first,
−
    -webkit-transform: translateY(-40px);
+
#second,
−
    -ms-transform: translateY(-40px);
+
#third,
−
    opacity: 1;
+
#fourth,
−
    visibility: visible;
+
#fifth,
 +
#sixth,
 +
#seventh,
 +
#eightth {
 +
  left: -1000px;
 +
  -ms-transition: tranform 1s ease;
 +
  -webkit-transition: transform 1s ease;
 +
  transition: transform 1s ease;
 +
  font-size: 100%;
 
}
 
}
  
−
#multbind{
+
#first{left: 0;}
−
  left: 430px;
+
#third{z-index : -100;}
−
  top: 230px;
+
#sixth{z-index: -100;}
 +
#fifth{z-index: -100;}
 +
#fourth{z-index: -100}
 +
#seventh{z-index: -100;}
 +
#eightth{z-index: -100; left: 1000px}
 +
 
 +
.container{
 +
    position: relative;
 +
    height: 500px;
 +
    margin: 3% 0;
 +
    width: 1000px;
 
}
 
}
  
−
#multbind:hover + #multbind_label {
+
#buttonleft {
−
    transform: translateY(-40px);
+
  z-index: 200;
−
    -webkit-transform: translateY(-40px);
+
  position: absolute;
−
    -ms-transform: translateY(-40px);
+
  top: 190px;
−
    opacity: 1;
+
  left: 0;
−
    visibility: visible;
+
 
}
 
}
  
−
#salmonella{
+
#buttonright{
−
   left: 750px;
+
   z-index: 200;
−
   top: 230px;
+
  position: absolute;
 +
  right: 0;
 +
   top: 190px;
 
}
 
}
  
−
#salmonella:hover + #salmonella_label {
+
#buttonleft img,
−
    transform: translateY(-40px);
+
#buttonright img{
−
    -webkit-transform: translateY(-40px);
+
  width: 50px;
−
    -ms-transform: translateY(-40px);
+
−
    opacity: 1;
+
−
    visibility: visible;
+
 
}
 
}
  
−
.circle:hover{
+
#content-slider{
−
    transition: all linear 0.5s;
+
  position: absolute;
−
    -webkit-transition: all linear 0.5s;
+
  top:0;
−
    -ms-transition: all linear 0.5s;
+
  left:50px
−
    transform: translate(-25px, -25px);
+
−
    -webkit-transform: translate(-25px, -25px);
+
−
    -ms-transform: translate(-25px, -25px);
+
−
    width: 150px;
+
−
    height: 150px;
+
−
    border-radius: 100px;
+
 
}
 
}
  
−
/* ========== END: Style for Details-Boxes ================*/
 
  
−
 
+
#buttonright:hover,
−
/* ========== Style for Circles in animated box ========== */
+
#buttonleft:hover{
−
 
+
  cursor: pointer;
−
.circle{
+
  cursor: hand;
−
    width: 100px;
+
−
    height: 100px;
+
−
    border-radius: 50px;
+
−
    border: 3px solid white;
+
−
    position: absolute;
+
 
}
 
}
  
−
.link_image{
+
.container{
−
    position: relative;
+
  position: relative;
 
}
 
}
  
−
/* ========== END: Style for Circles in animated box ========== */
+
.artboard{
 +
    box-sizing: border-box;
 +
    height: 470px;
 +
    border-radius: 60px;
 +
    background-color: #FCFCFC;
 +
  box-shadow: 1px 1px 10px rgb(136, 136, 136);
 +
  margin: 5px;
 +
  border: 2px solid rgb(0, 81, 162);
 +
}
  
 +
.sliderimage{
 +
  float: right;
 +
  margin: 3% 70px 3% 25px;
 +
  width: 350px
 +
}
  
−
/* ========== Remove Explore the DiaCHIP on small devices =================*/
+
.slidertext{
 +
  margin: 3% 25px 0 65px;
 +
  width: 350px;
 +
  text-align: justify;
 +
  line-height: 1;
 +
  float: left;
 +
}
  
−
@media screen and (max-width: 1000px){
+
.slidertext h1{
−
    #explore_DiaCHIP{
+
−
        display: none;
+
−
    }
+
  
 
}
 
}
  
−
@media screen and (max-width: 600px){
+
.slidertext p{
 +
  margin-bottom: 0;
 +
}
  
−
    #anti_tetanus_results, #device_results, #agfp_results, #specific_surfaces,#own_mix{
+
.slidertext.indent{
−
      margin-top:20px;
+
  clear: both;
−
    }
+
  margin-left: 15%;
−
 
+
  margin-top: 0;
−
    .results_headline,
+
  width: 290px;
−
    .headline_left{
+
−
      font-size: 120%;
+
−
      text-align: left;
+
−
      margin-bottom: 10px;
+
−
      margin-top: 20px;
+
−
      line-height: 120%;
+
−
      width: 100%;
+
−
    }
+
 
}
 
}
−
/* ========== END: Remove Explore the DiaCHIP on small devices =================*/
 
  
−
.result_box{
+
.dummy-image{
−
   overflow: hidden;
+
   background-color: black;
 +
  height: 400px;
 +
  width: 350px;
 
}
 
}
  
 
</style>
 
</style>
  
−
<script type="text/javascript">
 
  
−
// JQUERY for Details-Boxes
+
<html>
  
−
$(document).ready(function() {
+
<script>
−
    function close_accordion_section() {
+
$(document).ready(function(){
−
        $('.details_box_trigger').removeClass('active');
+
−
        $('.details_boxes').slideUp(300).removeClass('open');
+
−
    }
+
  
−
     $('.details_box_trigger').click(function(e) {
+
     var distance = 2;
−
        // Grab current anchor value
+
    // see: http://atomicrobotdesign.com/blog/web-development/how-to-disable-a-button-until-a-jquery-animation-has-finished/ //
−
        var currentAttrValue = $(this).attr('href');
+
    // bind the button to the function, so it can be unbound in the code and the button isnt clickable any more //
−
+
    $("#buttonleft").bind("click", moveleft);
−
        if($(e.target).is('.active')) {
+
  function moveleft(e) {
−
            close_accordion_section();
+
    // unbind the button //
−
        }else {
+
    $("#buttonleft").unbind();
−
            close_accordion_section();
+
    console.log("Click "+distance);
−
+
−
            // Add active class to section title
+
−
            $(this).addClass('active');
+
−
            // Open up the hidden content panel
+
−
            $(currentAttrValue).slideDown(300).addClass('open');  
+
−
        }
+
−
+
−
        e.preventDefault();
+
−
    });
+
  
−
     $('.closeButton').click(function(){
+
     if (distance == 1) {
−
          close_accordion_section();
+
      console.log("Distance: "+distance);
−
    });
+
      $('#first').css({"transform": "translateX(0)", "-webkit-transform": "translateX(0)", "-ms-transform": "translateX(0)"}); /* 2 first */
 +
        $('#second').css({"z-index": "100"}); /* begin second */
 +
        $('#third').css({});
 +
        $('#fourth').css({});
 +
        $('#fifth').css({});
 +
        $('#sixth').css({});
 +
        $('#seventh').css({"z-index": "-100", "transform": "translateX(0)", "-webkit-transform": "translateX(0)", "-ms-transform": "translateX(0)"}); /* 4 seventh */
 +
        $('#eightth').css({"transform": "translateX(0)", "-webkit-transform": "translateX(0)", "-ms-transform": "translateX(0)"}); /* 3 eightth */
 +
        distance = 2;
 +
    }
  
−
     $(window).resize(function(){
+
     else if (distance ==2) {
−
       console.log('window resized');
+
       console.log("Distance: "+distance);
−
       var scalefactor = $(window).width()/1000;
+
       $('#first').css({"transform": "translateX(1000px)", "-webkit-transform": "translateX(1000px)", "-ms-transform": "translateX(1000px)"}); /* 3 first */
−
      if ($(window).width() < 1000) {
+
         $('#second').css({"transform": "translateX(1000px)", "-webkit-transform": "translateX(1000px)", "-ms-transform": "translateX(1000px)"}); /* 2 second */
−
        console.log('scaling with factor'+scalefactor);
+
         $('#third').css({"z-index": "100"}); /* begin third */
−
        $('#Diagnosis').css({"transform":"scale("+scalefactor+")", "-webkit-transform":"scale("+scalefactor+")", "-ms-transform":"scale("+scalefactor+")"});
+
        $('#fourth').css({});
−
         $('#Assembling').css({"transform":"scale("+scalefactor+")", "-webkit-transform":"scale("+scalefactor+")", "-ms-transform":"scale("+scalefactor+")"});
+
        $('#fifth').css({});
−
      } else {
+
         $('#sixth').css({});
−
         $('#Diagnosis').css({"transform":"scale(1)", "-webkit-transform":"scale(1)", "-ms-transform":"scale(1)"});
+
        $('#seventh').css({});
−
         $('#Assembling').css({"transform":"scale(1)", "-webkit-transform":"scale(1)", "-ms-transform":"scale(1)"});
+
        $('#eightth').css({"z-index": "-100", "transform": "translateX(-2000px)", "-webkit-transform": "translateX(-2000px)", "-ms-transform": "translateX(-2000px)"}); /* 4 eightth */
−
      }
+
        distance = 3;
−
     })
+
     }
  
−
});
+
    else if (distance ==3) {
 +
      console.log("Distance: "+distance);
 +
      $('#first').css({"z-index": "-100", "transform": "translateX(-1000px)", "-webkit-transform": "translateX(-1000px)", "-ms-transform": "translateX(-1000px)", "-webkit-transform": "translateX(-1000px)", "-ms-transform": "translateX(-1000px)"}); /* 4 first */
 +
        $('#second').css({"transform": "translateX(2000px)", "-webkit-transform": "translateX(2000px)", "-ms-transform": "translateX(2000px)"}); /* 3 second */
 +
        $('#third').css({"transform": "translateX(1000px)", "-webkit-transform": "translateX(1000px)", "-ms-transform": "translateX(1000px)"}); /* 2 third */
 +
        $('#fourth').css({"z-index": "100"}); /* begin fourth */
 +
        $('#fifth').css({});
 +
        $('#sixth').css({});
 +
        $('#seventh').css({});
 +
        $('#eightth').css({});
 +
        distance = 4;
 +
    }
  
−
</script>
+
    else if (distance ==4) {
 +
      console.log("Distance: "+distance);
 +
      $('#first').css({});
 +
        $('#second').css({"z-index": "-100", "transform": "translateX(0)", "-webkit-transform": "translateX(0)", "-ms-transform": "translateX(0)"}); /* 4 second */
 +
        $('#third').css({"transform": "translateX(2000px)", "-webkit-transform": "translateX(2000px)", "-ms-transform": "translateX(2000px)"}); /* 3 third */
 +
        $('#fourth').css({"transform": "translateX(1000px)", "-webkit-transform": "translateX(1000px)", "-ms-transform": "translateX(1000px)"}); /* 2 fourth */
 +
        $('#fifth').css({"z-index": "100"}); /* begin fifth */
 +
        $('#sixth').css({});
 +
        $('#seventh').css({});
 +
        $('#eightth').css({});
 +
        distance = 5;
 +
    }
  
−
<div class="kommentar">
+
    else if (distance ==5) {
−
mehr Formulierungen wie: we demonstrated..., we (successfully) tested, we achieved...
+
      console.log("Distance: "+distance);
−
</div>
+
      $('#first').css({});
 +
        $('#second').css({});
 +
        $('#third').css({"z-index": "-100", "transform": "translateX(0)", "-webkit-transform": "translateX(0)", "-ms-transform": "translateX(0)"}); /* 4 third */
 +
        $('#fourth').css({"transform": "translateX(2000px)", "-webkit-transform": "translateX(2000px)", "-ms-transform": "translateX(2000px)"}); /* 3 fourth */
 +
        $('#fifth').css({"transform": "translateX(1000px)", "-webkit-transform": "translateX(1000px)", "-ms-transform": "translateX(1000px)"}); /* 2 fifth */
 +
        $('#sixth').css({"z-index": "100"}); /* begin sixth */
 +
        $('#seventh').css({});
 +
        $('#eightth').css({});
 +
        distance = 6;
 +
    }
  
−
<div class="content_box">
+
    else if (distance == 6) {
 +
      console.log("Distance: "+distance);
 +
      $('#first').css({});
 +
        $('#second').css({});
 +
        $('#third').css({});
 +
        $('#fourth').css({"z-index" : "-100", "transform": "translateX(0)", "-webkit-transform": "translateX(0)", "-ms-transform": "translateX(0)"}); /* 4 fourth */
 +
        $('#fifth').css({"transform": "translateX(2000px)", "-webkit-transform": "translateX(2000px)", "-ms-transform": "translateX(2000px)"}); /* 3 fifth */
 +
        $('#sixth').css({"transform": "translateX(1000px)", "-webkit-transform": "translateX(1000px)", "-ms-transform": "translateX(1000px)"}); /* 2 sixth */
 +
        $('#seventh').css({"z-index": "100"}); /* begin seventh */
 +
        $('#eightth').css({});
 +
        distance = 7;
 +
    }
  
−
  <div class="result_box">
+
     else if (distance == 7) {
−
     <div class="results_headline">Essential Results</div>
+
      console.log("Distance: "+distance);
−
    <p>
+
       $('#first').css({});
−
       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.
+
        $('#second').css({});
−
      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.
+
        $('#third').css({});
−
      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.
+
        $('#fourth').css({});
−
    </p>
+
        $('#fifth').css({"z-index": "-100", "transform": "translateX(0)", "-webkit-transform": "translateX(0)", "-ms-transform": "translateX(0)"}); /* 4 fifth */
−
  </div>
+
        $('#sixth').css({"transform": "translateX(2000px)", "-webkit-transform": "translateX(2000px)", "-ms-transform": "translateX(2000px)"}); /* 3 sixth */
 +
        $('#seventh').css({"transform": "translateX(1000px)", "-webkit-transform": "translateX(1000px)", "-ms-transform": "translateX(1000px)"}); /* 2 seventh */
 +
        $('#eightth').css({"z-index": "100"}); /* begin eightth */
 +
        distance = 8;
 +
    }
  
−
  <div class="result_box" id="anti-tetanus_results">
+
    else if (distance == 8) {
−
    <div class="results_headline headline_left">Detection of Anti-Tetanus Antibodies in Human Blood Serum</div>
+
      console.log("Distance: "+distance);
−
    <div class="image_box right">
+
      $('#first').css({"z-index": "100"}); /* begin first */
−
         <div class="thumb2 trien" style="width:250px">
+
        $('#second').css({});
−
          <div class="thumbinner">
+
         $('#third').css({});
−
            <img src="https://static.igem.org/mediawiki/2015/7/74/Freiburg_labjournal-roi_selection_20150817_429.png"></img>
+
        $('#fourth').css({});
−
            <div class="thumbcaption">
+
        $('#fifth').css({});
−
            Video 1: Detection of anti-tetanus antibodies in human serum.
+
        $('#sixth').css({"z-index": "-100", "transform": "translateX(0)", "-webkit-transform": "translateX(0)", "-ms-transform": "translateX(0)"}); /* 4 sixth */
−
            </div>
+
         $('#seventh').css({"transform": "translateX(2000px)", "-webkit-transform": "translateX(2000px)", "-ms-transform": "translateX(2000px)"}); /* 3 seventh */
−
          </div>
+
        $('#eightth').css({"transform": "translateX(-1000px)", "-webkit-transform": "translateX(-1000px)", "-ms-transform": "translateX(-1000px)"}); /* 2 eightth */
−
         </div>
+
        distance = 1;
−
    </div>
+
    }
−
    <p>
+
−
      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.
+
−
      <a class="details_box_trigger" href="#details_box-1">▼ Detailed Description.</a>
+
−
    </p>
+
−
  </div>
+
  
−
  <div id="details_box-1" class="details_boxes">
+
    // see: http://stackoverflow.com/questions/1836105/how-to-wait-5-seconds-with-jquery //
 +
    // wait until css-animation has finished. time is specified in css-transition property //
 +
    setTimeout(function(){
 +
      $("#buttonleft").bind("click", moveleft);
 +
    }, 1000);
 +
  }
  
−
    <div class="image_box left">
 
−
        <div class="thumb2 trien" style="width:250px"><div class="thumbinner">
 
−
            <img src="https://static.igem.org/mediawiki/2015/5/5b/Freiburg_tetanus_westernblot_protpur.png" style="width:230px"></img>
 
−
            <div class="thumbcaption">
 
−
            <strong>Figure 1: Western Blot analysis of purified tetanus antigen.</strong>
 
−
            </div>
 
−
        </div>
 
−
        </div>
 
−
    </div>
 
  
−
     <div class="image_box right">
+
     $("#buttonright").bind("click", moveright);
−
        <div class="thumb2 trien" style="width:250px"><div class="thumbinner">
+
  function moveright(e) {
−
            <img src="https://static.igem.org/mediawiki/2015/b/b9/Freiburg_tetanus%28-%29_quotion_picture.png" style="width:230px"></img>
+
−
            <div class="thumbcaption">
+
−
            <strong>Figure 2: Quotient picture of iRIf measurement of human serum sample taken before vaccination.</strong>
+
−
            </div>
+
−
        </div>
+
−
        </div>
+
−
    </div>
+
  
−
     <p>
+
     $("#buttonright").unbind();
−
      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>
+
     console.log("Click "+distance);
−
     </p>
+
  
−
     <p>
+
     if (distance == 1) {
−
      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.
+
       $('#first').css({"z-index": "-100"}); /* 1 second */
−
       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.
+
        $('#second').css({});  
−
     </p>
+
        $('#third').css({});
 +
        $('#fourth').css({});
 +
        $('#fifth').css({});
 +
        $('#sixth').css({"transform": "translateX(2000px)", "-webkit-transform": "translateX(2000px)", "-ms-transform": "translateX(2000px)"}); /* 6 third */
 +
        $('#seventh').css({"z-index": "100","transform": "translateX(1000px)", "-webkit-transform": "translateX(1000px)", "-ms-transform": "translateX(1000px)"}); /* 7 third */
 +
        $('#eightth').css({"transform": "translateX(-2000px)", "-webkit-transform": "translateX(-2000px)", "-ms-transform": "translateX(-2000px)"}); /* 8 first */
 +
        distance = 8;
 +
     }
  
 +
    else if (distance ==2) {
 +
      console.log("Distance: "+distance);
 +
      $('#first').css({"transform": "translateX(-1000px)", "-webkit-transform": "translateX(-1000px)", "-ms-transform": "translateX(-1000px)"}); /* 1 first */
 +
        $('#second').css({"z-index": "-100"}); /* 2 second */
 +
        $('#third').css({});
 +
        $('#fourth').css({});
 +
        $('#fifth').css({});
 +
        $('#sixth').css({});
 +
        $('#seventh').css({"transform": "translateX(2000px)", "-webkit-transform": "translateX(2000px)", "-ms-transform": "translateX(2000px)"}); /* 7 third */
 +
        $('#eightth').css({"z-index": "100","transform": "translateX(-1000px)", "-webkit-transform": "translateX(-1000px)", "-ms-transform": "translateX(-1000px)"}); /* 8 fourth */
 +
        distance = 1;
 +
    }
  
−
        <div class="image_box left">
+
    else if (distance ==3) {
−
            <div class="thumb2 trien" style="width:250px">
+
      console.log("Distance: "+distance);
−
              <div class="thumbinner">
+
      $('#first').css({"z-index": "100","transform": "translateX(0)", "-webkit-transform": "translateX(0)", "-ms-transform": "translateX(0)"}); /* 1 fourth */
−
                  <img src="https://static.igem.org/mediawiki/2015/f/f0/Freiburg_labjournal-surchem-20150902_elisa.png" style="width:230px"></img>
+
        $('#second').css({"transform": "translateX(0)", "-webkit-transform": "translateX(0)", "-ms-transform": "translateX(0)"}); /* 2 first */
−
                  <div class="thumbcaption">
+
        $('#third').css({"z-index": "-100"}); /* 3 second */
−
                  <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>
+
        $('#fourth').css({});
−
                  </div>
+
        $('#fifth').css({});
−
              </div>
+
        $('#sixth').css({});
−
            </div>
+
        $('#seventh').css({});
−
        </div>
+
        $('#eightth').css({"transform": "translateX(0)", "-webkit-transform": "translateX(0)", "-ms-transform": "translateX(0)"}); /* 8 third */
 +
        distance = 2;
 +
    }
  
−
     <p>
+
     else if (distance ==4) {
−
        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>.  
+
      console.log("Distance: "+distance);
−
     </p>
+
      $('#first').css({"transform": "translateX(1000px)", "-webkit-transform": "translateX(1000px)", "-ms-transform": "translateX(1000px)"}); /* 1 third */
 +
        $('#second').css({"z-index": "100","transform": "translateX(1000px)", "-webkit-transform": "translateX(1000px)", "-ms-transform": "translateX(1000px)"}); /* 2 fourth */
 +
        $('#third').css({"transform": "translateX(0)", "-webkit-transform": "translateX(0)", "-ms-transform": "translateX(0)"}); /*3 first */
 +
        $('#fourth').css({"z-index": "-100"}); /* 4 second*/
 +
        $('#fifth').css({});
 +
        $('#sixth').css({});
 +
        $('#seventh').css({});
 +
        $('#eightth').css({});
 +
        distance = 3;
 +
     }
  
−
        <div class="image_box right">
+
    else if (distance ==5) {
−
            <div class="thumb2 trien" style="width:250px"><div class="thumbinner">
+
      console.log("Distance: "+distance);
−
                <img src="https://static.igem.org/mediawiki/2015/2/26/Freiburg_tetanus%28%2B%29_quotion_picture.png" style="width:230px"></img>
+
      $('#first').css({});
−
                <div class="thumbcaption">
+
        $('#second').css({"transform": "translateX(2000px)", "-webkit-transform": "translateX(2000px)", "-ms-transform": "translateX(2000px)"}); /* 2 third */
−
                <strong>Figure 4: Quotient picture of iRIf measurement of human serum sample taken <b>three????</b> weeks after vaccination.</strong>
+
        $('#third').css({"z-index": "100","transform": "translateX(1000px)", "-webkit-transform": "translateX(1000px)", "-ms-transform": "translateX(1000px)"}); /* 3 fourth */
−
                </div>
+
        $('#fourth').css({"transform": "translateX(0)", "-webkit-transform": "translateX(0)", "-ms-transform": "translateX(0)"}); /* 4 first */
−
            </div>
+
        $('#fifth').css({"z-index": "-100"}); /* 5 second */
−
            </div>
+
        $('#sixth').css({});
−
         </div>
+
        $('#seventh').css({});
 +
        $('#eightth').css({});
 +
         distance = 4;
 +
    }
  
−
        <div class="image_box left">
+
    else if (distance == 6) {
−
            <div class="thumb2 trien" style="width:250px">
+
      console.log("Distance: "+distance);
−
              <div class="thumbinner">
+
      $('#first').css({});
−
                <img src="https://static.igem.org/mediawiki/2015/1/16/Freiburg_tetanus_binding_curve.png" style="width:230px">
+
        $('#second').css({});
−
              </img>
+
        $('#third').css({"transform": "translateX(2000px)", "-webkit-transform": "translateX(2000px)", "-ms-transform": "translateX(2000px)"}); /* 3 third */
−
                <div class="thumbcaption">
+
        $('#fourth').css({"z-index": "100","transform": "translateX(1000px)", "-webkit-transform": "translateX(1000px)", "-ms-transform": "translateX(1000px)"}); /* 4 fourth */
−
                <strong>PLATZHALTER! Figure 5: Binding curve of the tetanus measurements.</strong>
+
         $('#fifth').css({"transform": "translateX(0)", "-webkit-transform": "translateX(0)", "-ms-transform": "translateX(0)"}); /* 5 first */
−
                </div>
+
        $('#sixth').css({"z-index": "-100"}); /* 6 second */
−
            </div>
+
         $('#seventh').css({});
−
            </div>
+
        $('#eightth').css({});
−
         </div>
+
        distance =  5;
−
    <p>
+
    }
−
        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>
+
−
         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.  
+
  
−
         <br>
+
    else if (distance == 7) {
−
         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>.  
+
      console.log("Distance: "+distance);
−
     </p>
+
      $('#first').css({});
 +
         $('#second').css({});
 +
         $('#third').css({});
 +
        $('#fourth').css({"transform": "translateX(2000px)", "-webkit-transform": "translateX(2000px)", "-ms-transform": "translateX(2000px)"}); /* 4 third */
 +
        $('#fifth').css({"z-index": "100","transform": "translateX(1000px)", "-webkit-transform": "translateX(1000px)", "-ms-transform": "translateX(1000px)"}); /* 5 fourth */
 +
        $('#sixth').css({"transform": "translateX(0)", "-webkit-transform": "translateX(0)", "-ms-transform": "translateX(0)"}); /* 6 first */
 +
        $('#seventh').css({"z-index": "-100"}); /* 7 second */
 +
        $('#eightth').css({});
 +
        distance = 6;
 +
     }
  
−
     <div class="footnotes">
+
     else if (distance == 8) {
−
       <div class="fn"><sup><a class="fn_bot" href="#fnt__1" id="fn__1" name="fn__1">1)</a></sup>
+
      console.log("Distance: "+distance);
−
         <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>
+
       $('#first').css({});
−
      </div>
+
        $('#second').css({});
−
     </div>
+
        $('#third').css({});
 +
        $('#fourth').css({});
 +
        $('#fifth').css({"transform": "translateX(2000px)", "-webkit-transform": "translateX(2000px)", "-ms-transform": "translateX(2000px)"}); /* 5 third */
 +
         $('#sixth').css({"z-index": "100","transform": "translateX(1000px)", "-webkit-transform": "translateX(1000px)", "-ms-transform": "translateX(1000px)"}); /* 6 fourth */
 +
        $('#seventh').css({"transform": "translateX(0)", "-webkit-transform": "translateX(0)", "-ms-transform": "translateX(0)"}); /* 7 first */
 +
        $('#eightth').css({"z-index": "-100"}); /* 8 second */
 +
        distance = 7;
 +
     }
  
−
     <a class="closeButton" href="#anti_tetanus_results">▲ Close Box</a>
+
     setTimeout(function(){
−
   </div>
+
      $("#buttonright").bind("click", moveright);
 +
    }, 1000);
 +
   }
 +
});
 +
</script>
  
−
  <div class="result_box" id="agfp_results">
+
<div class="todo_box">
−
    <div class="kommentar">
+
expand text s.t. it leads to system overview <br>
−
      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
+
write appropriate content for slider: should present main subpoints of project in short sentence and link to them
−
      <br>
+
</div>
−
      Auch, dass euer Mix low-cost ist, und trotzdem auf dem Niveau (oder besser) eines kommerziellen kits liegt - Community gedanke etc.  (ps12092015)
+
−
    </div>
+
  
−
    <div class="results_headline">
+
<div class="header_box">  
−
        Detection of Anti-GFP Antibodies Using Cell-Free Expressed GFP on Ni-NTA
+
<h1>The DiaCHIP - A Versatile Detection System</h1>
−
    </div>
+
</div>
  
−
    <div class="image_box left">
+
<div class="image_box left" style="margin-bottom:10%;margin-left:-8%;margin-top:-10%;margin-right:-10%">
−
      <div class="thumbinner"style="width:250px;">
+
  <img align="left" alt="DiaCHIP_Sabi" src="https://static.igem.org/mediawiki/2015/a/af/Freiburg_DiaCHIP_Sabi.png" height="1430px">
−
        <meta http-equiv="X-UA-Compatible" content="IE=Edge"/>
+
</div>
−
        <video width="250" height="250" poster="https://static.igem.org/mediawiki/2015/9/93/Freiburg_20150819_NiNTA_cellfree_antigfp_preview.png" controls>
+
−
          <source src="https://static.igem.org/mediawiki/2015/4/4a/Freiburg_20150819_NiNTA_cellfree_antigfp_WEB.mp4" type="video/mp4">
+
−
          Your browser does not support the HTML5 video tag.
+
−
        </video>
+
−
        <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>
+
−
      </div>
+
−
    </div>
+
  
−
    <p>
+
<div style="width:120%;margin-bottom:22%">
−
      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.  
+
<div class="content_box3" style="margin-right:20%;margin-left:30%">
−
      <a class="details_box_trigger" href="#details_box-2">▼ Detailed Description.</a>
+
<h2>Project Motivation</h2>
−
    </p>
+
<p>
−
  </div>
+
        Serological tests are a key element in modern medicine. Especially for detection and identification of infectious diseases, performing several blood tests is inevitable. Testing for more than one disease at a time or diagnosing a patient with diffuse symptoms usually requires more than one serological test. Every single test that is performed increases the time of waiting for a result as well as the bill. In case of dangerous infectious diseases every minute until the onset of the appropriate treatment is important for life. What if there was a possibility to combine all this testing in one single chip which is affordable for everyone?
 +
</p>
 +
</div>
  
−
  <div id="details_box-2" class="details_boxes">
+
<div class="content_box3" style="margin-left:38%;margin-right:12%">
−
    <p>
+
<h2>Detecting Antigen-Antibody Interactions</h2>
−
      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.
+
<p>
−
      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).
+
  The DiaCHIP is an innovative tool to screen for a broad range of antibodies present in serum. Antibodies serve as indicator for an immune response towards an infectious diseases or a successful vaccination. They also play an important role in the diagnosis of autoimmune diseases. Identifying diseases by detecting disease associated antibodies in a patient's serum is an established method in  <a href="https://2015.igem.org/Team:Freiburg/Design" title="diagnostics_today">modern diagnostics</a>. <br>
−
      The spots were incubated on the slide over night.
+
Based on the very same principle, the DiaCHIP enables to simultaneously screen for multiple diseases at time, thereby reducing time and cost of a diagnosis. Especially the ability to differentiate between life threatening diseases and mild infections within a short time bears the potential to save lives.
−
    </p>
+
</p>
 +
</div>
  
−
    <div class="image_box left">
+
<div class="content_box3" style="margin-right:25%">
−
      <div class="thumb2 trien" style="width:250px">
+
<h2>The Concept</h2>
−
        <div class="thumbinner">
+
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  The key feature of the DiaCHIP concept is the combination of on-demand protein synthesis of disease related antigens and a novel method for label-free detection - all this packed into one device. The idea is to overcome challenges commonly found in protein array production and preservation. In addition, results can be obtained in a time- and cost-efficient manner; with a device simple enough to be rebuilt by future iGEM Teams.
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            <strong>Figure 1: Specific Ni-NTA surface on iRIf glass slide.</strong>
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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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      This step was followed by the <a href="https://2015.igem.org/Team:Freiburg/Protocols/iRIf_Measurement">blocking and washing protocol</a> to prevent further unspecific binding.
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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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            <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.
 
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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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        Building Our Low-Cost DiaCHIP Measuring Device
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                <h1>The DiaCHIP - System Overview</h1>
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                <p>The core of our new diagnostic device are two slides that form a microfluidic chamber. Therein an antigen array can be generated on demand on the bottom slide. By flushing the chamber with a blood sample, binding of antibodies present in the sample is detected with the optical detection method iRIf in real-time.</p>
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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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      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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        Establishing a Highly Specific Ni-NTA Surface
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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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      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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                <h1>(2)Communicating science</h1>
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                <p>Diagnosing diseases fast and reliable is not only an issue among medical staff, it is also subject to public interest. This has lead us to ask for people's opinions regarding the DiaCHIP. Although the method is based on synthetic biology, which is a problematic term for the broad public according to a survey initiated by the Leopoldina (National academy of science), we received a lot of positive feedback. </p>
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        Producing Our Cell-free Expression Mix
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                <p> Want to read <a href="https://2015.igem.org/Team:Freiburg/Practices">more</a>?</p>
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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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                <h1>(3)Modeling cell-free expression</h1>
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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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                <p>In order to optimize the DiaCHIP for future applications, we optimized the process of cell-free expression and diffusion over time. Making use of xxx parameters and xxx ordinary differential equations, we computed the size of the resulting antigen spots on the protein array and identified the factors limiting cell-free expression in the DiaCHIP. </p>
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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>(4)Measuring our blood</h1>
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                <p>One of the most promising results was obtained from the detection of anti-tetanus antibodies in human blood serum. The DiaCHIP analysis made it possible for us to distinguish serum samples from a team member before and after vaccination. Samples taken two weeks after vaccination produced higher signals, compared to those prior to antigen exposure.</p>
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      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!
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                <p> Want to read <a href="https://2015.igem.org/Team:Freiburg/Results">more</a>?</p>
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          <div class="hovertext" id="immobilization_label">DNA Immobilization</div>
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                <h1>(5)DNA Engineering</h1>
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                <p>Genetic fusion of different antigens and tags is a really basic requirement of our project. To enable many people to work on our DNA constructs in parallel we designed a cloning strategy easy to follow and additionally easy to expand for further needs.
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                                <br>
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Read more about how we combined different cloning methods to reduce our efforts in <a href="https://2015.igem.org/Team:Freiburg/Methods/Cloning" class="wikilink1">DNA Engineering.</a>
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  <h2>Diagnosis of Antigens</h2>
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                <p> Want to read <a href="https://2015.igem.org/Team:Freiburg/Results">more?</a></p>
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                <h1>(6)Protein purification</h1>
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                <p>Protein expression in the DiaCHIP is mediated by cell-free expression. As this is an advanced method dependent on the optimization of many parameters, we got back to conventional protein purification in <i>E. coli</i> for being able to compare the results of both techniques. <br>
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<a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/Protein_Purification">Read more</a> about the overexpression and purification of several antigenic peptides. 
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  <p> Click on one of the images to get a further insight on how we build our DiaCHIP </p>
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                <p> Want to read <a href="https://2015.igem.org/Team:Freiburg/Results">more?</a></p>
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                <h1>(7)Surface chemistry</h1>
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                <p>The production of a protein microarray in the DiaCHIP is based on specific immobilization of antigenic peptides on a glass slide. Therefore, we optimized a protocol for a specific surface chemistry until the proportion of unspecific binding of non-target proteins was reduced to a minimum.<br>
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<a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/Surface_Chemistry">Read more</a> about the different systems we tested on our way to high specificity.
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                <p> Want to read <a href="https://2015.igem.org/Team:Freiburg/Results">more?</a></p>
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                <h1>(8)Optical detection</h1>
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                <p>One disadvantage of currently available diagnostic test is the need for secondary labels that allow the detection of disease markers. Making use of an optical method based on the interference of light, the DiaCHIP can detect specific binding events on a protein microarray without further labeling. <br>
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<a class=”wikilink1” href=”https://2015.igem.org/Team:Freiburg/Project/iRIf>Read more</a> about this innovative tool and the physics behind it.
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                <p> Want to read <a href="https://2015.igem.org/Team:Freiburg/Results">more?</a></p>
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Revision as of 12:56, 17 September 2015

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The DiaCHIP - A Versatile Detection System

DiaCHIP_Sabi

Project Motivation

Serological tests are a key element in modern medicine. Especially for detection and identification of infectious diseases, performing several blood tests is inevitable. Testing for more than one disease at a time or diagnosing a patient with diffuse symptoms usually requires more than one serological test. Every single test that is performed increases the time of waiting for a result as well as the bill. In case of dangerous infectious diseases every minute until the onset of the appropriate treatment is important for life. What if there was a possibility to combine all this testing in one single chip which is affordable for everyone?

Detecting Antigen-Antibody Interactions

The DiaCHIP is an innovative tool to screen for a broad range of antibodies present in serum. Antibodies serve as indicator for an immune response towards an infectious diseases or a successful vaccination. They also play an important role in the diagnosis of autoimmune diseases. Identifying diseases by detecting disease associated antibodies in a patient's serum is an established method in modern diagnostics.
Based on the very same principle, the DiaCHIP enables to simultaneously screen for multiple diseases at time, thereby reducing time and cost of a diagnosis. Especially the ability to differentiate between life threatening diseases and mild infections within a short time bears the potential to save lives.

The Concept

The key feature of the DiaCHIP concept is the combination of on-demand protein synthesis of disease related antigens and a novel method for label-free detection - all this packed into one device. The idea is to overcome challenges commonly found in protein array production and preservation. In addition, results can be obtained in a time- and cost-efficient manner; with a device simple enough to be rebuilt by future iGEM Teams.

  • The DiaCHIP - System Overview

    The core of our new diagnostic device are two slides that form a microfluidic chamber. Therein an antigen array can be generated on demand on the bottom slide. By flushing the chamber with a blood sample, binding of antibodies present in the sample is detected with the optical detection method iRIf in real-time.

    Step by Step Overview

  • (2)Communicating science

    Diagnosing diseases fast and reliable is not only an issue among medical staff, it is also subject to public interest. This has lead us to ask for people's opinions regarding the DiaCHIP. Although the method is based on synthetic biology, which is a problematic term for the broad public according to a survey initiated by the Leopoldina (National academy of science), we received a lot of positive feedback.

    Want to read more?

  • (3)Modeling cell-free expression

    In order to optimize the DiaCHIP for future applications, we optimized the process of cell-free expression and diffusion over time. Making use of xxx parameters and xxx ordinary differential equations, we computed the size of the resulting antigen spots on the protein array and identified the factors limiting cell-free expression in the DiaCHIP.

    Want to read more?

  • (4)Measuring our blood

    One of the most promising results was obtained from the detection of anti-tetanus antibodies in human blood serum. The DiaCHIP analysis made it possible for us to distinguish serum samples from a team member before and after vaccination. Samples taken two weeks after vaccination produced higher signals, compared to those prior to antigen exposure.

    Want to read more?

  • (5)DNA Engineering

    Genetic fusion of different antigens and tags is a really basic requirement of our project. To enable many people to work on our DNA constructs in parallel we designed a cloning strategy easy to follow and additionally easy to expand for further needs.
    Read more about how we combined different cloning methods to reduce our efforts in DNA Engineering.

    Want to read more?

  • (6)Protein purification

    Protein expression in the DiaCHIP is mediated by cell-free expression. As this is an advanced method dependent on the optimization of many parameters, we got back to conventional protein purification in E. coli for being able to compare the results of both techniques.
    Read more about the overexpression and purification of several antigenic peptides.

    Want to read more?

  • (7)Surface chemistry

    The production of a protein microarray in the DiaCHIP is based on specific immobilization of antigenic peptides on a glass slide. Therefore, we optimized a protocol for a specific surface chemistry until the proportion of unspecific binding of non-target proteins was reduced to a minimum.
    Read more about the different systems we tested on our way to high specificity.

    Want to read more?

  • (8)Optical detection

    One disadvantage of currently available diagnostic test is the need for secondary labels that allow the detection of disease markers. Making use of an optical method based on the interference of light, the DiaCHIP can detect specific binding events on a protein microarray without further labeling.
    Read more about this innovative tool and the physics behind it.

    Want to read more?