Difference between revisions of "Team:Freiburg/Results/Immobilization"

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<h1 class="sectionedit1">Immobilization of DNA on PDMS</h1>
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<h1 class="sectionedit1">Cell-free expression of immobilized DNA</h1>
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<div class="level1">
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<p>
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An important part of the DiaCHIP is the possibility to ship and store information encoded in DNA to produce protein-arrays on demand. Therefore DNA has first to be fixed on silicon slides that later form the upper side of the microfluidic chamber. Using cell-free expression mix, the DNA can then be transcribed and translated into tagged proteins. These can bind the respective catcher on the glass slide forming the lower part of the chamber.
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</p>
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<h3 class="sectionedit2">Immobilizing DNA on a PDMS surface</h3>
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<div class="image_box left">
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<div class="thumb2 trien" style="width:250px"><div class="thumbinner">
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<a class="lightbox_trigger" href="hhttps://static.igem.org/mediawiki/2015/a/a3/Freiburg_files-20150903_pditc_surface.png">
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            <img src="https://static.igem.org/mediawiki/2015/a/a3/Freiburg_files-20150903_pditc_surface.png"></img>
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            <strong>Figure 1: Schematic of the APTES/PDITC surface. </strong>
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<h3 class="sectionedit2">Establishing a specific PDMS surface</h3>
 
 
<p>
 
<p>
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Before the DNA can be spotted onto the PDMS slide, the PDMS surface has to be prepared in a specialized manner (link protocol).
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Immobilizing DNA on a surface may be carried out the same way as immobilizing proteins, provided the DNA constructs contain an amino-group.
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To allow binding of a amio-tagged DNA a PDITC layer is coated (?) on top of the PDMS slide.
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Therefore DNA templates were amplified with PCR using an amino-labeled 3'-primer and a Cy3-labeled 5'-primer. The latter allows for detection of the bound DNA on the surface using an appropriate microarrays scanner
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To show the correct amplification of our constructs, an agarose-gel analysis was performed, confirming the right size of the DNA sequences (Figure 1).
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As DNA has to be fixed on a flow chamber consisting of the silicon PDMS (Polydimethylsiloxane), this silicon is first activated using <a href="https://2015.igem.org/Team:Freiburg/Project/Surface_Chemistry">oxygen plasma</a>. Coupling of DNA is achieved using the <a href="https://2015.igem.org/Team:Freiburg/Project/Surface_Chemistry">cross-linker PDITC </a> after coupling the silane APTES to the silicon.
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(Bild: layers)
 
(Bild: layers)
 
</p>
 
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<h3 class="sectionedit2">PCR amplification of DNA with Cy3-labelled primer and amino-labelled primer</h3>
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<div class="image_box right">
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<p>
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<div class="thumb2 trien" style="width:250px"><div class="thumbinner">
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To bind the DNA on the PDMS surface it has to be fused to a amino tag on the 3'-end. The amino-tag can interact with the PDITC-PDMS surface.
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<a class="lightbox_trigger" href="https://static.igem.org/mediawiki/2015/thumb/f/f5/Freiburg_spotting_microarrayscanner.png/735px-Freiburg_spotting_microarrayscanner.png">
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            <img src="https://static.igem.org/mediawiki/2015/thumb/f/f5/Freiburg_spotting_microarrayscanner.png/735px-Freiburg_spotting_microarrayscanner.png"></img>
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A Cy3-label is fused on the 5'-end of the DNA, thereby allowing detection of the spotted DNA via Cy3 measuring.
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            </a>
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PCR amplification of the coding sequence of GFP with the above mentioned primers was succesful. To verify correct DNA amplification an analysis on an agarose-gel was performed (fig.1).
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        </div>
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            <strong>Figure 2: Spotting and immobilization of DNA onto PDMA slide.</strong> A: Top view on the slide indicating the used spotting pattern. B: Microarray scanner measurement of Cy3 fluorescence.
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<h3 class="sectionedit2">Spotting of DNA - measuring of Cy3 signal</h3>
 
 
<p>
 
<p>
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DNA was spotted onto the prepared PDMS slide with a concentration of 25ng/µl. The slide was subsequently incubated over night and the DNA-solution was dried afterwards at 60°C. After washing of the slide (?) the DNA could be detected on the slide via measuring of the Cy3 signal.
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Coupling of DNA to the PDMS slide was achieved usign a DNA concentration of 25ng/µl spotted directly onto the slide (Figure 2, a). The slide was subsequently incubated over night and the DNA-solution was dried afterwards at 60°C. After washing the slide binding was confirmed by measuring the Cy3 fluorescence in a microarray scanner (Figure 2, b). The resulting fluorescence pattern clearly corresponds to the spotting pattern on the slide thereby confirming that the spotted DNA is responsible for the fluorescence signal.  
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Bild: Cy3-DNA on PDMS
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</p>
 
</p>
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<h3 class="sectionedit2">Cell-free expression of GFP from spotted DNA</h3>
 
<h3 class="sectionedit2">Cell-free expression of GFP from spotted DNA</h3>
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<p>
 
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A second method to verify binding of the DNA to the PDMS slide is cell-free expression of the DNA in a microfluidic chamber. DNA was spotted on the PDMS slide as described above and cell-free expression mix was flushed into the chamber. The slide was incubated with the cell-free expression mix for 2 hours, during which GFP was expressed.
 
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We could show successful expression of GFP from the spotted DNA using a fluorescence microscope.
 
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Bild: GFP fluorescence
 
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Text
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<div class="thumb2 trien" style="width:250px"><div class="thumbinner">
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<a class="lightbox_trigger" href="https://static.igem.org/mediawiki/2015/thumb/9/99/Freiburg_20150911_DNA_on_PDMS_7.0_oven30min_expr30min.jpg/794px-Freiburg_20150911_DNA_on_PDMS_7.0_oven30min_expr30min.jpg">
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            <img src="https://static.igem.org/mediawiki/2015/thumb/9/99/Freiburg_20150911_DNA_on_PDMS_7.0_oven30min_expr30min.jpg/794px-Freiburg_20150911_DNA_on_PDMS_7.0_oven30min_expr30min.jpg"></img>
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            </a>
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            <strong>Figure 3: Cell-free expressed GFP confirmed by fluorescence microscopy</strong>
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        </div>
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</div>
  
 
<p>
 
<p>
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</br>
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To confirm, that DNA was not only bound to the PDMS slide but is also suited for cell-free expression, we flushed the microfluidic chamber described above with cell-free mix. After incubation for two hours at room temperature the expressed GFP could be detected using a standard fluorescence microscope (Figure 3).
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<a href="https://2015.igem.org/Team:Freiburg/Methods/Cloning">More details about vector design and cloning strategies can be found here</a>.  
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More details on vector design and cloning strategies to generate the needed DNA can be found <a href="https://2015.igem.org/Team:Freiburg/Methods/Cloning"> here</a>.  
 
</p>
 
</p>
  

Revision as of 20:19, 15 September 2015

""

Sabine kannst du das bitte korrigieren usw. Ich hab einfach mal versucht was zu schreiben, aber ich hab echt nicht so viel Plan ^^ (LS 13/9)

Cell-free expression of immobilized DNA

An important part of the DiaCHIP is the possibility to ship and store information encoded in DNA to produce protein-arrays on demand. Therefore DNA has first to be fixed on silicon slides that later form the upper side of the microfluidic chamber. Using cell-free expression mix, the DNA can then be transcribed and translated into tagged proteins. These can bind the respective catcher on the glass slide forming the lower part of the chamber.

Immobilizing DNA on a PDMS surface

Figure 1: Schematic of the APTES/PDITC surface.

Immobilizing DNA on a surface may be carried out the same way as immobilizing proteins, provided the DNA constructs contain an amino-group. Therefore DNA templates were amplified with PCR using an amino-labeled 3'-primer and a Cy3-labeled 5'-primer. The latter allows for detection of the bound DNA on the surface using an appropriate microarrays scanner To show the correct amplification of our constructs, an agarose-gel analysis was performed, confirming the right size of the DNA sequences (Figure 1). As DNA has to be fixed on a flow chamber consisting of the silicon PDMS (Polydimethylsiloxane), this silicon is first activated using oxygen plasma. Coupling of DNA is achieved using the cross-linker PDITC after coupling the silane APTES to the silicon. (Bild: layers)

Figure 2: Spotting and immobilization of DNA onto PDMA slide. A: Top view on the slide indicating the used spotting pattern. B: Microarray scanner measurement of Cy3 fluorescence.

Coupling of DNA to the PDMS slide was achieved usign a DNA concentration of 25ng/µl spotted directly onto the slide (Figure 2, a). The slide was subsequently incubated over night and the DNA-solution was dried afterwards at 60°C. After washing the slide binding was confirmed by measuring the Cy3 fluorescence in a microarray scanner (Figure 2, b). The resulting fluorescence pattern clearly corresponds to the spotting pattern on the slide thereby confirming that the spotted DNA is responsible for the fluorescence signal.

Cell-free expression of GFP from spotted DNA

Figure 3: Cell-free expressed GFP confirmed by fluorescence microscopy

To confirm, that DNA was not only bound to the PDMS slide but is also suited for cell-free expression, we flushed the microfluidic chamber described above with cell-free mix. After incubation for two hours at room temperature the expressed GFP could be detected using a standard fluorescence microscope (Figure 3). More details on vector design and cloning strategies to generate the needed DNA can be found here.