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| − | <!-- SUBHEADER
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| − | ================================================== -->
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| − | <a id="link1"></a>
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| − | <div class="row">
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| − | <!-- CONTENT
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| − | <b></b><a href="#menu-toggle" class="btn btn-default" id="menu-toggle">Toggle Menu</a></p>
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| − | <script>$("#menu-toggle").click(function(e) {
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| − | <div class="row">
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| − | <!-- PROJECT DESCRIPTION-->
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| − | <div class="twelve columns">
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| − | </div>
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| − | <p>
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| − | In order to attempt to achieve the goals that we set ourselves we came up with three experiments to test our theories.
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| − | </p>
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| − | <p>
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| − | <H3>Experiment 1: Establishing Anchor Protein</H3>
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| − | <br>1. Take a briobrick part (JO4450) and transform it into a chemically competent cell (Top 10)
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| − | <br>2. Grow cells overnight then miniprep them to extract the transformed plasmid
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| − | <br>3. Double digestion of plasmid to cut out the RFP gene, leaving only the plasmid backbone
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| − | <br>4. PCR the full construct G-Block (containing Zif268 and Lpp_OmpA) and digest the product with the corresponding enzymes
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| − | <br>5. <a href="http://synbio.tsl.ac.uk/golden-gate-assembly-protocol/">Golden gate assembly</a> of plasmid backbones
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| − | and full construct.
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| − | <br>6. Transform electrocompetent E. <i>coli</i> cells with full plasmid
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| − | <br>7.Grow cells
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| − | <br>8. Miniprep full construct plasmid from modified cells
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| − | <br>9. Digest plasmid with enzymes matching the cut sites around the Lpp_OmpA gene
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| − | <br>10. PCR the rest of the anchor proteins from the G-blocks ordered (INP, PGSA, and BCLA)
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| − | <br>11. Ligate the anchor proteins into digested full construct plasmid
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| − | <br>12. Transform all plasmids into electrocompetent E. <i>coli</i> - End product is 4 different types of cell, each expressing the full construct with slightly different transmembrane domains
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| − | <br>13. Prepare transformed cells for microscopy (mount cells on glass slides)
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| − | <br>14. Bind antibodies to FLAG tag - measure under fluorescent microscope
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| − | <br>15. Determine whether the gene is being expressed and whether the expressed protein is being transported and is binding to the membrane of the E. <i>coli</i>
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| − | <br>16. Compare intensities - select anchor protein that fluoresces brightest, this anchor protein with be used for all subsequent experiments.
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| − | </p>
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| − | <p>
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| − | <br> <H3>Experiment 2: Establishing Zinc Finger DNA Binding Proteins</H3>
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| − | <br>1. Golden gate assembly of plasmid backbones and many G-blocks (one of each candidate zinc finger protein and the winning anchor protein) and with fluorescent protein code
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| − | <br>2. Transform E. Coli with plasmid
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| − | <br>3. Make wells of cognate DNA of the zinc fingers, introduce transformed E. Coli to the plate, leave to bind, wash plate and compare fluorescence of the E. Coli cells to determine which zinc finger binds strongest
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| − | <br>4. Select 4 zinc fingers which bind strongest to their cognate DNA
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| − | <br>Ben models = requires time points, do at the same time
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| − | <br>Microscopy - do with both alive and dead
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| − | <br>Test both coverslip or on slide
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| − | </p>
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| − | <p>
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| − | <H3>Experiment 3: Checking Colour</H3>
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| − | <br>1. Transform E. Coli with each colour fluorescent protein and the anchor protein and each zinc finger protein
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| − | - YFP, GFP, RFP - need to select a blue fluorescent protein
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| − | <br>2. Test whether simple images can be made - green circle on a red square
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| − | <br>3. Test colour mixing - mixing fluorescent cells to make colours not already present
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| − | <br>4. Test complex images
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| − | </p>
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| − | </div>
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| − | </div>
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| − | <!-- end main content-->
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| − | </html>
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| − | {{WarwickFooter}}
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