Difference between revisions of "Team:Freiburg/Results"

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<div class="content_box">
 
<div class="content_box">
  
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<h1 class="sectionedit1">Assembling the DiaCHIP</h1>
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<div id="anti_tetanus_results">
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<div class="level1">
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<div class="floatbox right">
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<h2>Intro</h2>
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<h2>
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<p>
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We successfully diagnosed anti-Tetanus antibodies in human blood serum
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The following section summarizes the most interesting results we obtained this summer. All together we were able to establish a cell-free expression system that can be used to copy a DNA immobilized on a PDMS slide. The expressed protein is then immobilized on another slide via a specific tag system resulting in a distinct pattern. A microfluidic system is used to flush the slide with an antibody solution. Specific antibody-antigen interactions were successfully detected by imaging reflectometric interference (iRIf), a label-free detection method. For diagnostic applications, the immobilized proteins are antigenic peptides specific for a certain pathogen.
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</h2>
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</p>
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<p>
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<h2>DNA on PDMS</h2>
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Check out this video showing how the antibodies in the serum bind our tetanus antigens on the DiaChip
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<div class="image_box left">
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</p>
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<div class="thumb2 trien" style="width:310px"><div class="thumbinner"><a class="lightbox_trigger" href="https://static.igem.org/mediawiki/2015/2/2e/Freiburg_2015_freiburg_dna_on_pdms.png"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/2/2e/Freiburg_2015_freiburg_dna_on_pdms.png" width="300"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=results_overview&amp;media=2015_freiburg_dna_on_pdms.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div><strong>Figure 1: DNA immobilization on activated PDMS.</strong> PCR amplification of the expression cassette was performed with an amino-labelled reverse primer and a Cy3-labelled forward primer. Immobilized DNA is visualized by Cy3 fluorescence.</div></div></div></div>
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<div class="image_box right">
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<div>
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<img src="https://static.igem.org/mediawiki/2015/7/74/Freiburg_labjournal-roi_selection_20150817_429.png" width="100%"></img>
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<p>
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</div>
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The first step in the production process of the DiaCHIP was to genetically fuse antigen coding sequences to a 10xHis Tag that was used for surface immobilization later. The whole expression cassette including promoter and terminator regions was amplified by PCR using an amino-labelled reverse primer. Via this amino group, the DNA was immobilized on an activated PDMS surface. The forward primer used for this PCR was labelled with Cy3. As it is shown in figure 1, spotting the DNA on the activated surface resulted in a distinct pattern visualized by Cy3 fluorescence.
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</p>
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<h2>Cell-free expression</h2>
 
  
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<p>
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<div class="floatbox left">
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To establish a cell-free expression system, a bacterial lysate was produced and supplemented with several energy sources as well as co-factors and ions. GFP expression in our system was compared to a commercially available kit. In figure 2, the relative fluorescence of the samples is shown over time. After two hours the fluorescence indicates a x fold change of the amount of GFP in the sample compared to the beginning of the reaction. The commercially available kit reaches an x fold increase in relative fluorescence.
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<p>
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<br/>
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We took a persons blood before and after a tetanus vaccination and screened for a tetanus infection.
 +
An immunized person has antibodies against tetanus antigens - we bound spots of our purified tetanus antigens onto the specific surface of our chip. We then incubated the DiaChip with 33 µl of the blood serum for half an hour and measured the results.
  
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Additionally, it was shown that the expressed GFP is not only functional in terms of fluorescence but it also exhibits the same binding affinity to a commercial anti-GFP antibody as conventionally purified GFP (figure 3). Thus, our cell-free expression system can be used to mediate the copying process from a DNA template to a protein microarray.
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Take a look at the video and see for yourself how good our DiaChip performed.
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</p>
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<div class="flexbox">
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And remember: that this isn’t an ELISA, we don’t need no expensive secondary Antibodies!
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<div class="thumb2 trien" style="width:410px"><div class="thumbinner"><a class="lightbox_trigger" href="https://static.igem.org/mediawiki/2015/5/50/Freiburg_2015_freiburg_cellfex_gfp_over_time.png"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/5/50/Freiburg_2015_freiburg_cellfex_gfp_over_time.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_over_time.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div><strong>Figure 2: Comparison with self-made cell-free expression system and a commercially available kit.</strong> The relative fluorescence of cell-free expressed GFP is monitored over 2 hours in comparison with a commercial kit. For each system a no-DNA control was added enabling to calculate the fold change. </div></div></div><div class="thumb2 trien" style="width:310px"><div class="thumbinner">Invalid Link<div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=results_overview&amp;media=figure_name.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div><strong>Figure 3: Western Blot of conventionally and cell-free expressed GFP.</strong> </div></div></div>
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</p>
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</div>
 
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<h2>Specific surface</h2>
 
  
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<p>
 
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Using a cell-free expression system requires the establishment of a specific surface that prevents the binding of non-target proteins. A stable protocol for the production of Ni-NTA surfaces was developed. The 10xHis Tag complexes nickel ions on the surface resulting in a sufficiently strong binding of the protein. In figure 4, an unspecific PDITC surface and a Ni-NTA surface are compared. Flushing the slides with the same antibody solution resulted in a weaker signal for PDITC, which is explained by the binding of many non-target proteins blocking the surface for the His-tagged protein.
 
−
<br/>
 
  
−
Additionally, we have shown that cell-free expressed and therefore non-purified protein can be efficiently immobilized on the surface in a sufficient amount to detect antibody binding by iRIf (figure 5).
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<div id="agfp_results">
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Figure 6 shows that several different antigen spots can be distinguished by flushing the slide with antibodies that are specifically binding to just one of them. Only the spot where the antigen corresponding to the used antibody is immobilized exhibits a signal.
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<div class="floatbox right">
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</p>
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<h2>
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<div class="flexbox">
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We measured the binding of anti-GFP to cell-free expressed GFP in blood serum
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<div class="thumb2 trien" style="width:310px"><div class="thumbinner">Invalid Link<div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=results_overview&amp;media=figure_name2.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div><strong>Figure 4: Quotion picture of an iRIf meassurement of non-purified, His-tagged GFP(?) on PDITC compared to Ni-NTA.</strong> Whole cell lysate was spotted either on an unspecific PDITC surface (A) or on a specific Ni-NTA surface (B) and flushed with anti-GFP(?). The quotion picture shows the change in thickness at distinct spots related to the rest of the surface.</div></div></div><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 5: Binding curve of anti-GFP to cell-free expressed GFP on a Ni-NTA surface.</strong> Cell-free expressed GFP was spotted on a specific Ni-NTA surface and flushed with anti-GFP. The binding curve indicated a binding event a certain spot.</div></div></div>
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</h2>
 +
<p>
 +
We expressed GFP with our own cell-free expression system and bound it to the surface of our DiaChip. We than ran some rabbit blood serum over the chip which we had spiked with anti-GFP antibodies. The results show that our cell-free proteins can be measured even in a complex solution such as blood serum. </p>
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<div class="floatbox left">
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<img src="https://static.igem.org/mediawiki/2015/7/74/Freiburg_labjournal-roi_selection_20150817_429.png" width="100%"></img>
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<h2>Diagnostics</h2>
 
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<p>
 
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These results can be used to establish a diagnostic device based on the detection of specific antibodies in a patient’s blood sample. We were able to express a specific antigen for <em>Salmonella</em> Typhimurium and immobilize it on the surface. Flushing the slide with a self-purified single chain variable fragment specifically binding to the antigen resulted in strong signal at the <em>Salmonella</em> spot, whereas no signal was seen at the negative control spot (figure 7).
 
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<br/>
 
  
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Another antigen that was expressed is a specific marker for <em>Clostridium tetani</em>. We obtained serum samples of a person before and after vaccination against <em>C. tetani</em> and showed that the DiaCHIP can be used to detect antibodies against the bacterium in the positive serum sample. In contrast, no binding could be observed in the negative sample (figure 8).
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<div id="device_results">
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<h1>
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We built our very own, low-cost DiaChip device - and can measure with it!
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</h1>
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<div class="flexbox">
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<img src="https://static.igem.org/mediawiki/2015/7/74/Freiburg_labjournal-roi_selection_20150817_429.png" width="80%"></img>
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</div>
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<p>
 +
A green LED, two lenses and a regular SLR Camera is basically all you need to build your own iRIf device.
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</p>
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</div>
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<div class="content_box">
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<div class="flexbox">
 
<div class="flexbox">
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<div class="thumb2 trien" style="width:410px"><div class="thumbinner"><a class="lightbox_trigger" href="https://static.igem.org/mediawiki/2015/2/2f/Freiburg_2015_freiburg_salmonella_binding_quotion.png"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/2/2f/Freiburg_2015_freiburg_salmonella_binding_quotion.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_salmonella_binding_quotion.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div><strong>Figure 7: iRIf meassurement of spotted <em>Salmonella</em> Typhimurium antigen.</strong> The self-purified single chain variable fragment was used to detect an antigen specific for <em>Salmonella</em> Typhimurim spotted on a Ni-NTA surface. A: Binding curve. B: Quotion picture.</div></div></div><div class="thumb2 trien" style="width:410px"><div class="thumbinner"><a href="https://static.igem.org/mediawiki/2015/6/63/Freiburg_2015_freiburg_tetanus_binding_quotion.png" class="lightbox_trigger"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/6/63/Freiburg_2015_freiburg_tetanus_binding_quotion.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_tetanus_binding_quotion.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div><strong>Figure 8: iRIf meassurement of spotted, cell-free expressed <em>C. tetani</em> antigen.</strong> Cell-free expressed tetanus antigen was spotted on a Ni-NTA surface and flushed with a human serum sample before (A and B) and after vaccination (C and D). A, C: binding curves. B, D: quotion pictures.</div></div></div>
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<a href="https://2015.igem.org/Team:Freiburg/Results/Assembling">
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<img src="https://static.igem.org/mediawiki/2015/0/0f/Freiburg_homepage_chip_blood.png" width="100%">
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<a href="https://2015.igem.org/Team:Freiburg/Results/Diagnostics">
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<img src="https://static.igem.org/mediawiki/2015/0/0f/Freiburg_homepage_chip_blood.png" width="100%">
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</a>
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</div>
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<h2>Conclusion</h2>
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<p> Click on one of the images to get further insight how we build up our DiaCHIP </p>
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<p>
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All in all, we established a method for the production of a protein microarray by copying a DNA template combined with label-free detection of antibody binding events. We showed that DNA can be immobilized on activated PDMS and serve as a template for cell-free expression. Cell-free expressed protein is immobilized on another surface via a specific tag system. Additionally, we were able to distinguish several immunologically relevant antigen spots by specific antibody binding out of a human serum sample.
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Revision as of 17:57, 7 September 2015

""

We successfully diagnosed anti-Tetanus antibodies in human blood serum

Check out this video showing how the antibodies in the serum bind our tetanus antigens on the DiaChip

We took a persons blood before and after a tetanus vaccination and screened for a tetanus infection. An immunized person has antibodies against tetanus antigens - we bound spots of our purified tetanus antigens onto the specific surface of our chip. We then incubated the DiaChip with 33 µl of the blood serum for half an hour and measured the results. Take a look at the video and see for yourself how good our DiaChip performed. And remember: that this isn’t an ELISA, we don’t need no expensive secondary Antibodies!

We measured the binding of anti-GFP to cell-free expressed GFP in blood serum

We expressed GFP with our own cell-free expression system and bound it to the surface of our DiaChip. We than ran some rabbit blood serum over the chip which we had spiked with anti-GFP antibodies. The results show that our cell-free proteins can be measured even in a complex solution such as blood serum.

We built our very own, low-cost DiaChip device - and can measure with it!

A green LED, two lenses and a regular SLR Camera is basically all you need to build your own iRIf device.

Click on one of the images to get further insight how we build up our DiaCHIP