Difference between revisions of "Team:Warwick/Protocols"

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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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<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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<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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<br>Expected results:
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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>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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<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>
 +
<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>
 +
Expected results:
 +
<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>
 +
<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>
 +
</ul>
 
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Revision as of 14:32, 17 September 2015

Warwick iGEM 2015

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