Difference between revisions of "Team:Warwick/Cloning"

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<h4>Binding Affinity Modelling</h4>
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<h4>Cloning and Transformation</h4></div>
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Our project relied greatly on previously established techniques for cloning modified genes into plasmids and transforming those plasmids into competent cells. We wanted to make our part as modular and easy to change as possible, meaning we had to rely heavily on techniques such as Gibson assembly to construct our plasmid. By offering our part with changeable parts we hoped to make our part more useful for further research, and allow for deeper applications than the ones we initially thought of.
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Having interchangeable zinc fingers allows the diversity of the cells that can be glued together using our system to be much greater by allowing each different cell type or individual cell to have a specific DNA sequence that it binds to, so that fine control of spatial positioning of cells can be achieved.
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By having modular anchor domains, we allow for the expression of the zinc finger on the surface of the cell to be controlled as well. By changing the anchor domain, you affect the number of zinc fingers expressed on the surface of the cell, as well as their binding affinity, due to different anchors forcing the zinc fingers closer or further away from the cell. Our design also includes unique cut sites for the linkers in the part, so that they too can be cut out and changed for longer or shorter ones, also affecting zinc finger expression or binding.
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<img src="https://static.igem.org/mediawiki/2015/1/1d/Warwick_Modularity.png" align="middle" height="950px" width="950px">
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<p>This diagram shows our starting point in the project with the JO4450 biobrick part expressing RFP. It also shows the modularity in our design with the interchangeable anchor domains and zinc finger domains. </p>
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<H3>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 <i>E.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 <i>E.coli</i> - end product: four 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 <i>E.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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<img src="https://static.igem.org/mediawiki/2015/4/45/Warwick_Diagram_of_anchor_domain_modularity.jpeg" align="middle">
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<br> <H3>Establishing Zinc Finger DNA Binding Proteins</H3>
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<br>1. Miniprep full construct plasmid from the first experiment
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<br>2. Digest plasmid with enzymes corresponding to the cut sites around the Zif268 gene
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<br>3. PCR zinc finger genes from the G-blocks ordered
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<br>4. Golden gate assembly of digested full construct plasmid and the three zinc finger PCR products
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<br>5. Transform electrocompetent <i>E.coli</i> with each modified plasmid - end product: four different types of cells, each with the same anchor protein but different zinc finger DNA binding domains
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<br>6. Prepare the cells for microscopy by binding them to slides
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<br>7. Prepare four different sets of fluorescent oligos that each contain the target sequence for a zinc finger
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<br>8. Under a fluorescent microscope, wash the fixed cells with the fluorescent oligos, allow them to bind, then wash off the excess non-binded oligos
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<br>9. Measure fluorescence of the cells - cells that bind the fluorescent oligos on the surface will glow, allowing us to see that the zinc fingers are binding the target DNA sequence
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<br>10. Compare measurements to the same cell types washed with non-target sequence fluorescent DNA - if the cells have similar fluorescence it means that the zinc fingers are binding DNA indiscriminately, against what they were designed to do
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<br>11. Given that the zinc fingers bind DNA and discriminate between target and non-target DNA, select the cells that bind target DNA the best and grow these up
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<img src="https://static.igem.org/mediawiki/2015/2/2c/Warwick_Diagram_of_zinc_finger_modularity.jpeg" align="middle">
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<H3> Making it Coloured</H3>
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<br>1. Make the selected E. <i>coli</i> cells that express strong DNA binding zinc fingers from experiment 2 electro or chemically competent
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<br>2. Transform each E. <i>coli</i> type with a unique colour fluorescent protein that expresses intracellularly - end product: three/four different E. <i>coli</i> cell types, each expressing a different zinc finger and each glowing a different colour
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<br>3. Prepare slides by binding oligos with target DNA sequences to specific places on the slide to create a shape
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<br>4. Wash over the slide with complimentary E. <i>coli</i> cells expressing the cognate zinc finger and allow the cells to bind, then wash the excess cells off the slide
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<br>5. Use a fluorescent microscope to determine whether the cells that stuck to the slide are bound in the predetermined pattern, indicating that they have bound themselves to the oligos printed on the slide
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<br>6. Repeat steps 3-5 using multiple different oligos and washing over with multiple cells to create a multi-coloured pattern
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<br>7. Repeat with more complex patterns
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<img src="https://static.igem.org/mediawiki/2015/4/45/Warwick_Diagram_of_experiment_3.jpeg"  align="middle">
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Latest revision as of 17:51, 17 September 2015

Warwick iGEM 2015

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