Difference between revisions of "Team:Freiburg/Results"
Line 22: | Line 22: | ||
.image_box.left { | .image_box.left { | ||
− | + | margin-right: 50px; | |
} | } | ||
.image_box.right { | .image_box.right { | ||
− | + | margin-left: 50px; | |
} | } | ||
+ | |||
+ | |||
+ | /* ========== Style for Details-Boxes ================*/ | ||
+ | |||
+ | .details_box_trigger{ | ||
+ | display:inline-block; | ||
+ | transition:all linear 0.15s; | ||
+ | text-decoration: none !important; | ||
+ | color: #0051A2; | ||
+ | font-weight: 500; | ||
+ | float: right; | ||
+ | } | ||
+ | |||
+ | |||
+ | .details_box_trigger.active { | ||
+ | } | ||
+ | |||
+ | .details_boxes{ | ||
+ | padding:15px; | ||
+ | display: none; | ||
+ | overflow:hidden; | ||
+ | box-shadow:0px 1px 3px rgba(0,0,0,0.25); | ||
+ | border-radius:20px; | ||
+ | background:#EEE; | ||
+ | } | ||
+ | |||
+ | /* ========== END: Style for Details-Boxes ================*/ | ||
+ | |||
</style> | </style> | ||
+ | |||
+ | <script type="text/javascript"> | ||
+ | |||
+ | // JQUERY for Details-Boxes | ||
+ | |||
+ | $(document).ready(function() { | ||
+ | function close_accordion_section() { | ||
+ | $('.details_box_trigger').removeClass('active'); | ||
+ | $('.details_boxes').slideUp(300).removeClass('open'); | ||
+ | } | ||
+ | |||
+ | $('.details_box_trigger').click(function(e) { | ||
+ | // Grab current anchor value | ||
+ | var currentAttrValue = $(this).attr('href'); | ||
+ | |||
+ | if($(e.target).is('.active')) { | ||
+ | close_accordion_section(); | ||
+ | }else { | ||
+ | close_accordion_section(); | ||
+ | |||
+ | // Add active class to section title | ||
+ | $(this).addClass('active'); | ||
+ | // Open up the hidden content panel | ||
+ | $(currentAttrValue).slideDown(300).addClass('open'); | ||
+ | } | ||
+ | |||
+ | e.preventDefault(); | ||
+ | }); | ||
+ | }); | ||
+ | |||
+ | // Details-Boxes | ||
+ | |||
+ | </script> | ||
+ | |||
<div class="kommentar"> | <div class="kommentar"> | ||
Line 41: | Line 103: | ||
<div class="content_box"> | <div class="content_box"> | ||
+ | |||
+ | <h1>Main Results</h1> | ||
<div id="anti_tetanus_results"> | <div id="anti_tetanus_results"> | ||
− | + | <div class="kommentar"> | |
− | + | In den text eine bessere Reihenfolge rein bringen. Es wird vom blut gesprochen, dann Expression in E.coli und dann wieder Blut. </strong> | |
− | + | </div> | |
− | + | <h2>Detection of anti-Tetanus antibodies in human blood serum</h2> | |
− | + | <div class="image_box right"> | |
<div class="thumb2 trien" style="width:250px"><div class="thumbinner"> | <div class="thumb2 trien" style="width:250px"><div class="thumbinner"> | ||
− | + | <img src="https://static.igem.org/mediawiki/2015/7/74/Freiburg_labjournal-roi_selection_20150817_429.png"></img> | |
</div> | </div> | ||
<strong>Video 1: Detection of anti-tetanus antibodies in human serum.</strong> | <strong>Video 1: Detection of anti-tetanus antibodies in human serum.</strong> | ||
− | + | </div> | |
− | + | </div> | |
− | + | <p> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | <strong>We specifically detected anti-Tetanus antibodies in human blood serum.</strong> | |
− | + | 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 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. | |
− | + | ||
− | + | <a class="details_box_trigger" href="#details_box-1">Detailed description.</a> | |
− | + | </p> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
+ | <div id="details_box-1" class="details_boxes"> | ||
+ | <p> | ||
+ | Applying the sample taken three weeks after vaccination to the DiaCHIP resulted in a significant signal at the antigen spot, whereas no signal was obtained with the sample assumed to be negative. But convince yourself of how great the DiaCHIP performed. The video on the right shows these measurements right next to each other in real-time. | ||
+ | <br> | ||
+ | Besides tetanus, some other antigens of immunological relevance were taken into account. See all the <a href="https://2015.igem.org/Team:Freiburg/Results/Diagnostics">results we obtained in terms of diagnostics</a>, including an approach for quantification of the measurements. | ||
+ | </p> | ||
+ | </div> | ||
</div> | </div> | ||
− | |||
− | |||
<div id="agfp_results"> | <div id="agfp_results"> | ||
− | + | <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</div> | |
− | + | <h2> | |
− | + | Detection of anti-GFP antibodies in rabbit serum using cell-free expressed GFP | |
− | + | </h2> | |
− | + | <div class="image_box left"> | |
− | + | <div class="thumb2 trien" style="width:250px"><div class="thumbinner"> | |
− | + | <img src="https://static.igem.org/mediawiki/2015/7/74/Freiburg_labjournal-roi_selection_20150817_429.png"></img> | |
− | + | </div> | |
− | + | <strong>Figure 1: Binding of anti-GFP antibodies to cell-free expressed GFP.</strong> | |
− | + | </div> | |
− | + | </div> | |
− | + | <p> | |
− | + | To show that cell-free expressed proteins can be immobilized on the protein surface while maintaining their antibody binding properties, the expression mix was spotted on a specific Ni-NTA surface after GFP expression. Cell-free expressed GFP was spotted on a specific Ni-NTA slide and flushed with serum of a rabbit immunized against GFP. The lowest spot consisting of cell-free expressed GFP shows a significant signal compared to the negative control (middle spot). | |
− | + | <a class="details_box_trigger" href="#details_box-2">Detailed description.</a> | |
− | + | </p> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
+ | <div id="details_box-2" class="details_boxes"> | ||
+ | <p> | ||
+ | Serum of a rabbit immunized with GFP was applied to the DiaCHIP, which resulted in a strong signal at the referring spot evoked by antibody binding events (figure 1). Thus, His-tagged GFP was successfully expressed and immobilized on the surface, still enabling anti-GFP antibodies in a serum sample to bind. | ||
+ | <br> | ||
+ | Find out more about the <a href="https://2015.igem.org/Team:Freiburg/Results/Assembling">preparation of the DiaCHIP</a> by producing a protein microarray from a DNA template using cell-free expression. | ||
+ | </p> | ||
+ | </div> | ||
</div> | </div> | ||
− | |||
<div id="device_results"> | <div id="device_results"> | ||
− | + | <h1> | |
− | + | Building our very own, low-cost DiaCHIP measuring device. | |
− | + | </h1> | |
− | + | <div class="image_box right"> | |
− | + | <div class="thumb2 trien" style="width:250px"><div class="thumbinner"> | |
− | + | <a href="https://static.igem.org/mediawiki/2015/b/b7/Freiburg_Own_Device_Foto.jpg"> | |
− | + | <img src="https://static.igem.org/mediawiki/2015/2/2e/Freiburg_Own_Device_Foto_preview.jpg"></img> | |
− | + | </a> | |
− | + | </div> | |
− | + | <strong>Figure 2: Functional, but low-cost variant of the measuring device.</strong> | |
− | + | </div> | |
− | + | </div> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
+ | <p> | ||
+ | The measuring device we used in collaboration with the AG Roth (ZBSA) to detect antibody binding events is a really expensive machine, but the physics which it is based on is rather simple. Our self-built device consistent of not much more than two lenses and a camera (A) reliably detected the binding of anti-GFP to GFP (B). To enable future iGEM teams to profit from this label-free detection method as we did, a cost-efficient and easy to rebuild variant of the device was developed (figure 2A). | ||
+ | <a class="details_box_trigger" href="#details_box-3">Detailed description.</a> | ||
+ | </p> | ||
+ | |||
+ | <div id="details_box-3" class="details_boxes"> | ||
+ | <p> | ||
+ | Figure 2B shows the binding of anti-GFP to GFP as it was established before, measured with our own device. | ||
+ | Look here, to see how you can <a href="https://2015.igem.org/Team:Freiburg/Results/Own_Device">build your own device</a> for label-free detection of antibody binding on a protein microarray needing two lenses, a camera and a little bit of fine feeling (??) sensitivety/sensitiveness/finesse/sure instincts. | ||
+ | </p> | ||
+ | </div> | ||
+ | </div> | ||
<div id="specific_surfaces"> | <div id="specific_surfaces"> | ||
− | + | <h1> | |
− | + | Specific surfaces | |
− | + | </h1> | |
− | + | ||
− | + | <p> | |
− | + | For immobilization of the antigens on the chip we developed our own specific Ni-NTA surface. All expression constructs have a His-tag fusd to the coding sequence, resulting in antigens that can bind specifically to our surface. Compared to an unspecific surface (PDITC) we could show that this Ni-NTA surface allows accurate binding of target proteins and prevents unspecific binding from other proteins of the cell-free mix. | |
− | + | <a class="details_box_trigger" href="#details_box-4">Detailed description.</a> | |
− | + | </p> | |
− | + | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
+ | <div id="details_box-4" class="details_boxes"> | ||
+ | <p> | ||
+ | Subtext | ||
+ | </p> | ||
+ | </div> | ||
+ | </div> | ||
<div id="own_mix"> | <div id="own_mix"> | ||
− | + | <h1> | |
− | + | Own cell-free mix | |
− | + | </h1> | |
− | + | ||
− | + | <p> | |
− | + | The copying mechanism we are using to assemble a protein array from a DNA array template is based on cell-free expression. During our project we successfully established our own cell-free expression system from scratch, starting with an <em>E. coli</em> lysate. With our own cell-free mix we succeeded in expression of correctly folded GFP and luciferase. Comparison of the Dia-Mix with a other cell-free expression kits proved that our own cell-free expression mix is even better than a commercial kit! | |
− | + | <a class="details_box_trigger" href="#details_box-5">Detailed description.</a> | |
− | + | </p> | |
− | + | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
+ | <div id="details_box-5" class="details_boxes"> | ||
+ | <p> | ||
+ | Subtext | ||
+ | </p> | ||
+ | </div> | ||
+ | </div> | ||
</div> | </div> | ||
− | |||
− | |||
Revision as of 19:09, 12 September 2015
![](https://static.igem.org/mediawiki/2015/8/85/Freiburg_wiki_background_2_parted_left.png)
![](https://static.igem.org/mediawiki/2015/f/f4/Freiburg_wiki_background_2_parted_right.png)
Nicole: Hab ich es richtig verstanden, dass hier nur eure Highlights and Ergebnissen dargestellt werden sollen (mit verlinkung zu den anderen Ergebnissen)? Was haltet ihr von einer kurzen Einleitung... In the last months we (aimed to) developed a diagnostic tool which enables [...] We succeded to detect anti-tetanus antibodies in human serum, develop/generate our own cell-free mix...
mehr Formulierungen wie: we demonstrated..., we (successfully) tested, we achieved...
Main Results
Detection of anti-Tetanus antibodies in human blood serum
![](https://static.igem.org/mediawiki/2015/7/74/Freiburg_labjournal-roi_selection_20150817_429.png)
We specifically detected anti-Tetanus antibodies in human blood serum. 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 before and after vaccination and could directly detect its effect. To capture the antibodies, the corresponding antigen was expressed in E. coli, purified by His-tag affinity purification and spotted on a specific Ni-NTA surface. Detailed description.
Applying the sample taken three weeks after vaccination to the DiaCHIP resulted in a significant signal at the antigen spot, whereas no signal was obtained with the sample assumed to be negative. But convince yourself of how great the DiaCHIP performed. The video on the right shows these measurements right next to each other in real-time.
Besides tetanus, some other antigens of immunological relevance were taken into account. See all the results we obtained in terms of diagnostics, including an approach for quantification of the measurements.
Detection of anti-GFP antibodies in rabbit serum using cell-free expressed GFP
![](https://static.igem.org/mediawiki/2015/7/74/Freiburg_labjournal-roi_selection_20150817_429.png)
To show that cell-free expressed proteins can be immobilized on the protein surface while maintaining their antibody binding properties, the expression mix was spotted on a specific Ni-NTA surface after GFP expression. Cell-free expressed GFP was spotted on a specific Ni-NTA slide and flushed with serum of a rabbit immunized against GFP. The lowest spot consisting of cell-free expressed GFP shows a significant signal compared to the negative control (middle spot). Detailed description.
Serum of a rabbit immunized with GFP was applied to the DiaCHIP, which resulted in a strong signal at the referring spot evoked by antibody binding events (figure 1). Thus, His-tagged GFP was successfully expressed and immobilized on the surface, still enabling anti-GFP antibodies in a serum sample to bind.
Find out more about the preparation of the DiaCHIP by producing a protein microarray from a DNA template using cell-free expression.
Building our very own, low-cost DiaCHIP measuring device.
The measuring device we used in collaboration with the AG Roth (ZBSA) to detect antibody binding events is a really expensive machine, but the physics which it is based on is rather simple. Our self-built device consistent of not much more than two lenses and a camera (A) reliably detected the binding of anti-GFP to GFP (B). To enable future iGEM teams to profit from this label-free detection method as we did, a cost-efficient and easy to rebuild variant of the device was developed (figure 2A). Detailed description.
Figure 2B shows the binding of anti-GFP to GFP as it was established before, measured with our own device. Look here, to see how you can build your own device for label-free detection of antibody binding on a protein microarray needing two lenses, a camera and a little bit of fine feeling (??) sensitivety/sensitiveness/finesse/sure instincts.
Specific surfaces
For immobilization of the antigens on the chip we developed our own specific Ni-NTA surface. All expression constructs have a His-tag fusd to the coding sequence, resulting in antigens that can bind specifically to our surface. Compared to an unspecific surface (PDITC) we could show that this Ni-NTA surface allows accurate binding of target proteins and prevents unspecific binding from other proteins of the cell-free mix. Detailed description.
Subtext
Own cell-free mix
The copying mechanism we are using to assemble a protein array from a DNA array template is based on cell-free expression. During our project we successfully established our own cell-free expression system from scratch, starting with an E. coli lysate. With our own cell-free mix we succeeded in expression of correctly folded GFP and luciferase. Comparison of the Dia-Mix with a other cell-free expression kits proved that our own cell-free expression mix is even better than a commercial kit! Detailed description.
Subtext