Difference between revisions of "Team:Warwick/Project2"

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and full construct.
 
and full construct.
 
<br>6. Transform electrocompetent E. <i>coli</i> cells with full plasmid
 
<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>7. Grow cells
 
<br>8. Miniprep full construct plasmid from modified cells
 
<br>8. Miniprep full construct plasmid from modified cells
 
<br>9. Digest plasmid with enzymes matching the cut sites around the Lpp_OmpA gene
 
<br>9. Digest plasmid with enzymes matching the cut sites around the Lpp_OmpA gene
 
<br>10. PCR the rest of the anchor proteins from the G-blocks ordered (INP, PGSA, and BCLA)
 
<br>10. PCR the rest of the anchor proteins from the G-blocks ordered (INP, PGSA, and BCLA)
 
<br>11. Ligate the anchor proteins into digested full construct plasmid
 
<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>12. Transform all plasmids into electrocompetent E. <i>coli</i> - end product: four different types of cell, each expressing the full construct with slightly different transmembrane domains
 
<br>13. Prepare transformed cells for microscopy (mount cells on glass slides)  
 
<br>13. Prepare transformed cells for microscopy (mount cells on glass slides)  
 
<br>14. Bind antibodies to FLAG tag - measure under fluorescent microscope
 
<br>14. Bind antibodies to FLAG tag - measure under fluorescent microscope
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<br>16. Compare intensities - select anchor protein that fluoresces brightest, this anchor protein with be used for all subsequent experiments.
 
<br>16. Compare intensities - select anchor protein that fluoresces brightest, this anchor protein with be used for all subsequent experiments.
 
</p>
 
</p>
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<p>
 
<p>
 
<br> <H3>Experiment 2: Establishing Zinc Finger DNA Binding Proteins</H3>
 
<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>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 E. <i>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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<br>2. Transform E. Coli with plasmid
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</p>
  
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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>
 
 
<p>
 
<p>
 
<H3>Experiment 3: Checking Colour</H3>
 
<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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<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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- YFP, GFP, RFP - need to select a blue fluorescent protein
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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>2. Test whether simple images can be made - green circle on a red square
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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>3. Test colour mixing - mixing fluorescent cells to make colours not already present
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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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<br>4. Test complex images
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</p>
 
</p>

Revision as of 13:58, 9 September 2015

Warwick iGEM 2015

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