Difference between revisions of "Team:Warwick/Protocols"
| Line 29: | Line 29: | ||
<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. | <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 (<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> | + | <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 46: | Line 46: | ||
<p>________________________________________________________________________________________________________________________________________________</p> | <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> | ||
| − | + | 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> | <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> | ||
| − | <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> | + | <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 63: | Line 63: | ||
<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> | ||
<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> | <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. | |
| − | <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> | + | <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> | ||
Revision as of 11:42, 18 September 2015
