Difference between revisions of "Team:Warwick/Protocols"
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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> | <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> | ||
| − | Expected results: | + | <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> | <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> | <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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</p> | </p> | ||
| − | + | <ul> | |
<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> |
| − | < | + | <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> |
| − | < | + | <b>Expected results:</b> |
| − | < | + | <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> |
| − | < | + | <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> |
| − | < | + | <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> |
| − | < | + | <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> | ||
| − | + | </ul> | |
Revision as of 14:33, 17 September 2015