Difference between revisions of "Team:Warwick/Protocols"

 
(27 intermediate revisions by 3 users not shown)
Line 22: Line 22:
 
<h4>Experiments</h4>
 
<h4>Experiments</h4>
 
</div>
 
</div>
 +
<p>________________________________________________________________________________________________________________________________________________</p>
 
<p>  
 
<p>  
−
<h4>Experiment 1: Testing the binding of specifically designed DNA strands to glass        slides</h4>
 
  
 +
<h3>Experiment 1: Testing the binding of specifically designed DNA strands to glass slides</h3>
  
 +
<br>Objective: To ensure that our zinc finger binding domains are able to bind to a glass slide, and can be visualised through immunofluorescent microscopy.
 
<ul>
 
<ul>
−
<li>Glass slides were prepared <a href="https://static.igem.org/mediawiki/2015/f/f8/WarwickGlassSlideProtocol.pdf">Glass Slide Preperation Protocol</a> by being cleaned and functionalised (with HCl and GOPTS respectively).</li>
+
<br><li>Glass slides were prepared (<ahref="https://static.igem.org/mediawiki/2015/f/f8/WarwickGlassSlideProtocol.pdf">Glass Slide Preperation Protocol</a>)  by being cleaned and functionalised (with HCl and GOPTS respectively).</li>
 
<li>Specifically designed oligonucleotides containing zinc finger binding domains were introduced to the slides.</li>
 
<li>Specifically designed oligonucleotides containing zinc finger binding domains were introduced to the slides.</li>
 
<li>These oligonucleotides comprise of a general adaptor strand, a specific short strand and a specific long strand.</li>
 
<li>These oligonucleotides comprise of a general adaptor strand, a specific short strand and a specific long strand.</li>
Line 35: Line 37:
 
<li>Slides treated with GOPTS should show fluorescence.</li>
 
<li>Slides treated with GOPTS should show fluorescence.</li>
 
<li>Untreated cells should show no fluorescence.</li>
 
<li>Untreated cells should show no fluorescence.</li>
−
This is a control to ensure that our zinc finger binding domains can be bound to glass slides.
+
 
 
</ul>
 
</ul>
  
Line 42: Line 44:
  
 
<ul>
 
<ul>
−
 
+
<p>________________________________________________________________________________________________________________________________________________</p>
−
<h3> Experiment 2: Testing the expression of zinc finger proteins (on the surface of E. coli cells) upon induction with IPTG </h3>
+
<h3> Experiment 2: Testing the expression of zinc finger proteins on the surface of E. coli cells upon induction with IPTG </h3>
−
<li>We tested the extent to which each of our zinc fingers (zif 268, sZF2, sZF10 and sZF14) proteins were expressed on the surface of our cells by using immunofluorescence microscopy.</li>
+
Objective: To ensure that our plasmid is being translated into protein, folding correctly and then being transported to the cell membrane effectively. Through the binding of fluorescent antibodies to the cell surface, we should be able to visualise the cells.
 +
<p style="float: right;"> <img src="https://static.igem.org/mediawiki/2015/3/3d/Warwick_diagram_of_redirecting_protein.jpeg" height="500px" width="500px" border="50px"></p>
 +
<br><br><li>We tested the extent to which each of our zinc fingers (zif 268, sZF2, sZF10 and sZF14) proteins were expressed on the surface of our cells by using immunofluorescence microscopy.</li>
 
<li>To do this, a FLAG tag (predesigned to be within our construct) was fused to the surface display anchor proteins to which our zinc finger proteins are attached.</li>
 
<li>To do this, a FLAG tag (predesigned to be within our construct) was fused to the surface display anchor proteins to which our zinc finger proteins are attached.</li>
 
<li>The introduction of an anti-flag antibody, followed by a secondary antibody (a fluorescently labelled anti-mouse antibody) allowed our E. coli cells to be visualised. <a href="https://static.igem.org/mediawiki/2015/2/2e/WarwickiGEMBacterialProtocolUpdated.pdf">Bacterial Immunofluorescence Protocol</a>.</li>
 
<li>The introduction of an anti-flag antibody, followed by a secondary antibody (a fluorescently labelled anti-mouse antibody) allowed our E. coli cells to be visualised. <a href="https://static.igem.org/mediawiki/2015/2/2e/WarwickiGEMBacterialProtocolUpdated.pdf">Bacterial Immunofluorescence Protocol</a>.</li>
Line 54: Line 58:
  
 
<ul>
 
<ul>
 +
 +
<p>________________________________________________________________________________________________________________________________________________</p>
 
<p>
 
<p>
 
<H3> Experiment 3: Reciprocal experiment - binding of fluorescently labelled oligonucleotides to immobilised cells </H3>
 
<H3> Experiment 3: Reciprocal experiment - binding of fluorescently labelled oligonucleotides to immobilised cells </H3>
−
<li>E. coli cells expressing each of our 4 zinc finger proteins were immobilised onto glass slides <a href="https://static.igem.org/mediawiki/2015/b/b9/WarwickBacterialProtocolUpdated.pdf">Bacterial Immunofluorescence Protocol</a>.</li>
+
<p><p style="float: left;"> <img src="https://static.igem.org/mediawiki/2015/2/20/Warwick_Reciprocal_experiment.jpeg" height="500px" width="500px" border="50px"></p>
 +
Objective: To ensure that our zinc finger proteins are able to bind to fluorescently labelled oligonucleotides which contain the zinc finger binding domain.
 +
<br><br><li>E. coli cells expressing each of our 4 zinc finger proteins were immobilised onto glass slides <a href="https://static.igem.org/mediawiki/2015/b/b9/WarwickBacterialProtocolUpdated.pdf">Bacterial Immunofluorescence Protocol</a>.</li>
 
<li>Fluorescently labelled oligonucleotides (containing the zinc finger binding domains) were added.</li>
 
<li>Fluorescently labelled oligonucleotides (containing the zinc finger binding domains) were added.</li>
 
<li>Binding of the zinc finger proteins to the fluorescent oligonucleotides allows visualisation of the cells by immunofluorescence microscopy. </li>  
 
<li>Binding of the zinc finger proteins to the fluorescent oligonucleotides allows visualisation of the cells by immunofluorescence microscopy. </li>  
Line 67: Line 75:
  
 
</ul>
 
</ul>
 +
<br>
 +
<br>
 +
<br>
  
 +
<p>________________________________________________________________________________________________________________________________________________</p>
  
 
<b><H3> Future Experiments </H3></b>
 
<b><H3> Future Experiments </H3></b>
Line 73: Line 85:
 
</p><br>
 
</p><br>
 
<ul>
 
<ul>
 +
 
<p>
 
<p>
 
<H3> Experiment 4: Binding fluorescent zinc finger expressing cells to oligos on a glass slide </H3>
 
<H3> Experiment 4: Binding fluorescent zinc finger expressing cells to oligos on a glass slide </H3>
−
<li>A range of zinc finger expressing cells would be prepared (with the cells for each zinc finger corresponding to one particular colour).</li>
+
Objective: To see whether we can control the precise locations of different coloured cells through binding of the zinc finger proteins to oligonucleotides in specific positions on a glass slide.
 +
<br><br><li>A range of zinc finger expressing cells would be prepared (with the cells for each zinc finger corresponding to one particular colour).</li>
 
<li>Different oligos would be placed on a glass slide in specific places (either by hand, or using a parafilm template). The positioning of these oligos would determine where cells of any given colour are able to bind.
 
<li>Different oligos would be placed on a glass slide in specific places (either by hand, or using a parafilm template). The positioning of these oligos would determine where cells of any given colour are able to bind.
 
  </li>  
 
  </li>  
 
<li>Using immunofluorescence microscopy, we would visualise our slide, and see cells of different colours coming together to form a ‘picture’.
 
<li>Using immunofluorescence microscopy, we would visualise our slide, and see cells of different colours coming together to form a ‘picture’.
 +
<img src="https://static.igem.org/mediawiki/2015/0/00/Warwick_Diagram_of_specific_zinc_finger_DNA_binding.jpeg" border="50px">
 
</li>
 
</li>
  
Line 86: Line 101:
  
  
−
 
+
<p>________________________________________________________________________________________________________________________________________________</p>
  
  
Line 92: Line 107:
 
<p>
 
<p>
 
<H3> Experiment 5: Binding fluorescent zinc finger expressing cells to oligonucleotide adhesive </H3>
 
<H3> Experiment 5: Binding fluorescent zinc finger expressing cells to oligonucleotide adhesive </H3>
−
<li>Make more DNA origmami Y structures.</li>
+
Objective: To see whether we can control the binding of different cell types to an engineered DNA structure.
 +
 
 +
<br><br><li>Make more DNA origami Y structures.</li>
 
<li>Grow up three different types of zinc-finger expressing cells.
 
<li>Grow up three different types of zinc-finger expressing cells.
 
  </li>  
 
  </li>  
Line 99: Line 116:
 
<li>Using immunofluorescence microscopy, we would visualise our slide, and see cells of different colours coming together to form a ‘picture’.
 
<li>Using immunofluorescence microscopy, we would visualise our slide, and see cells of different colours coming together to form a ‘picture’.
 
</li>
 
</li>
−
 
+
<img style="-webkit-user-select: none" src="https://static.igem.org/mediawiki/2015/1/14/Warwick_Diagram_of_specific_zinc_finger_DNA_binding_2.jpeg">
  
 
</ul>
 
</ul>

Latest revision as of 18:15, 18 September 2015

Warwick iGEM 2015

Open Menu