Difference between revisions of "Team:Warwick/Protocols"

 
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<h4>Experiments</h4>
 
<h4>Experiments</h4>
 
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<p>________________________________________________________________________________________________________________________________________________</p>
 
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<p>  
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<h4>Experiment 1: Testing the binding of specifically designed DNA strands to glass        slides</h4>
 
  
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<h3>Experiment 1: Testing the binding of specifically designed DNA strands to glass slides</h3>
  
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<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>
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<br><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>
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<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>
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<br><li>Specifically designed oligonucleotides containing zinc finger binding domains were introduced to the slides.</li>
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<li>Specifically designed oligonucleotides containing zinc finger binding domains were introduced to the slides.</li>
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<br><li>These oligonucleotides comprise of a general adaptor strand, a specific short strand and a specific long strand.</li>
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<li>These oligonucleotides comprise of a general adaptor strand, a specific short strand and a specific long strand.</li>
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<br><li>Terminal amine groups within the oligonucleotides bind (by a nucleophilic addition reaction) to the epoxy group of GOPTS, sticking the DNA to the glass slides.</li>
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<li>Terminal amine groups within the oligonucleotides bind (by a nucleophilic addition reaction) to the epoxy group of GOPTS, sticking the DNA to the glass slides.</li>
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<br><li>The presence of an EcoR1 cut site in the oligonucleotide allows us to have an extra level of control in our experiments. Although the zinc finger proteins will stay attached to their binding domains, cutting the oligonucleotide at this site allows cells to become ‘unstuck’ from the slides.</li>
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<li>The presence of an EcoR1 cut site in the oligonucleotide allows us to have an extra level of control in our experiments. Although the zinc finger proteins will stay attached to their binding domains, cutting the oligonucleotide at this site allows cells to become ‘unstuck’ from the slides.</li>
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<br>Expected results:
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<b>Expected results:</b>
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<br><li>Slides treated with GOPTS should show fluorescence.</li>
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<li>Slides treated with GOPTS should show fluorescence.</li>
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<br><li>Untreated cells should show no fluorescence.</li>
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<li>Untreated cells should show no fluorescence.</li>
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<br>This is a control to ensure that our zinc finger binding domains can be bound to glass slides.
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</p>  
 
</p>  
  
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<h3> Experiment 2: Testing the expression of zinc finger proteins (on the surface of E. coli cells) upon induction with IPTG </h3>
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<ul>
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<br>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.
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<p>________________________________________________________________________________________________________________________________________________</p>
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<br>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.
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<h3> Experiment 2: Testing the expression of zinc finger proteins on the surface of E. coli cells upon induction with IPTG </h3>
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<br>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>.
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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.
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<br>Expected results:
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<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>
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<br>No / little fluorescence: Zinc finger proteins should not be expressed on the surface of the wild type and uninduced E. coli cells. This means that the primary antibody (and therefore the fluorescent secondary antibody) are unable to bind to the cell surface, so very little (or even no) fluorescence should be seen. Only background fluorescence should be seen.
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<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>
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<br>Fluorescence: Induced E. coli cells express the zinc fingers (and therefore the anchor protein) on their cell surface. This allows the primary and (subsequently) secondary antibody to bind, making our cells fluoresce.
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<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>
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<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>
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<b>Expected results:</b>
 +
<li>No / little fluorescence: Zinc finger proteins should not be expressed on the surface of the wild type and uninduced E. coli cells. This means that the primary antibody (and therefore the fluorescent secondary antibody) are unable to bind to the cell surface, so very little (or even no) fluorescence should be seen. Only background fluorescence should be seen.</li>
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<li>Fluorescence: Induced E. coli cells express the zinc fingers (and therefore the anchor protein) on their cell surface. This allows the primary and (subsequently) secondary antibody to bind, making our cells fluoresce.</li>
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</ul>
 
</p>
 
</p>
  
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<ul>
  
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<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>
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<br>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>.
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<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>
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<br>Fluorescently labelled oligonucleotides (containing the zinc finger binding domains) were added.
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Objective: To ensure that our zinc finger proteins are able to bind to fluorescently labelled oligonucleotides which contain the zinc finger binding domain.
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<br>Binding of the zinc finger proteins to the fluorescent oligonucleotides allows visualisation of the cells by immunofluorescence microscopy.
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<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>
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<br>Expected results:
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<li>Fluorescently labelled oligonucleotides (containing the zinc finger binding domains) were added.</li>
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<br>Immobilised wild type DH5α Z1 cells (washed with oligos) should show no fluorescence, as the oligos should not be able to bind to the cells.
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<li>Binding of the zinc finger proteins to the fluorescent oligonucleotides allows visualisation of the cells by immunofluorescence microscopy. </li>
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<br>Uninduced cells should not be expressing any zinc finger proteins on their surface, so should show no fluorescence. Any fluorescence seen could be due to a ‘leaky’ promoter.
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<b>Expected results:</b>
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<br>Cells that have been induced (with IPTG) and then washed with the corresponding oligos should show fluorescence. This is because the oligos should bind to the cells, so are not removed during the washing stages.
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<li>Immobilised wild type DH5α Z1 cells (washed with oligos) should show no fluorescence, as the oligos should not be able to bind to the cells.</li>
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<br>To test the specificity of our zinc finger proteins, each cell type was washed with oligos matching a different zinc finger. In this step, any fluorescence would suggest cross-reactivity between the zinc fingers and their binding domains.
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<li>Uninduced cells should not be expressing any zinc finger proteins on their surface, so should show no fluorescence. Any fluorescence seen could be due to a ‘leaky’ promoter.</li>
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<li>Cells that have been induced (with IPTG) and then washed with the corresponding oligos should show fluorescence. This is because the oligos should bind to the cells, so are not removed during the washing stages.</li>
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<li>To test the specificity of our zinc finger proteins, each cell type was washed with oligos matching a different zinc finger. In this step, any fluorescence would suggest cross-reactivity between the zinc fingers and their binding domains.</li>
 
</p>
 
</p>
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</ul>
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<br>
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<p>________________________________________________________________________________________________________________________________________________</p>
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<b><H3> Future Experiments </H3></b>
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<p>Due to time restrictions, we were unable to carry out the following experiment. This experiment involved ‘painting a microscopic picture’ with our fluorescent cells. This was to demonstrate specific localisation of cells controlled by our programmable Brixells.
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</p><br>
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<ul>
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<p>
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<H3> Experiment 4: Binding fluorescent zinc finger expressing cells to oligos on a glass slide </H3>
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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.
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<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>
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<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.
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</li>
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<li>Using immunofluorescence microscopy, we would visualise our slide, and see cells of different colours coming together to form a ‘picture’.
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<img src="https://static.igem.org/mediawiki/2015/0/00/Warwick_Diagram_of_specific_zinc_finger_DNA_binding.jpeg" border="50px">
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</li>
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</ul>
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<p>________________________________________________________________________________________________________________________________________________</p>
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<ul>
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<p>
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<H3> Experiment 5: Binding fluorescent zinc finger expressing cells to oligonucleotide adhesive </H3>
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Objective: To see whether we can control the binding of different cell types to an engineered DNA structure.
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<br><br><li>Make more DNA origami Y structures.</li>
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<li>Grow up three different types of zinc-finger expressing cells.
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</li>
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<li>Wash the cells and origami together, and allow to sit for 2-3 hours.
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</li>
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<li>Using immunofluorescence microscopy, we would visualise our slide, and see cells of different colours coming together to form a ‘picture’.
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</li>
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<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">
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</ul>
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Latest revision as of 18:15, 18 September 2015

Warwick iGEM 2015

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