Difference between revisions of "Team:Warwick/Protocols"

 
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<h4>Protocols</h4>
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<h4>Experiments</h4>
 
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<p>________________________________________________________________________________________________________________________________________________</p>
 
<p>  
 
<p>  
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<br> To ensure that our experiments went as smoothly and efficiently as possible, we followed tried and tested protocols.
 
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</p>
 
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<H3>Competent Cell Protocol</H3>
 
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<h5>Produces chemically competent cells</h5>
 
  
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<h3>Experiment 1: Testing the binding of specifically designed DNA strands to glass slides</h3>
  
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<br>IMPORTANT: Pre-cool all the buffers and tubes that you use to 4ºC – This
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<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.
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<ul>
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<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>
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<li>Specifically designed oligonucleotides containing zinc finger binding domains were introduced to the slides.</li>
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<li>These oligonucleotides comprise of a general adaptor strand, a specific short strand and a specific long strand.</li>
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<li>Terminal amine groups within the oligonucleotides bind (by a nucleophilic addition reaction) to the epoxy group of GOPTS, sticking the DNA to the glass slides.</li>
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<li>The presence of an EcoR1 cut site in the oligonucleotide allows us to have an extra level of control in our experiments. Although the zinc finger proteins will stay attached to their binding domains, cutting the oligonucleotide at this site allows cells to become ‘unstuck’ from the slides.</li>
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<b>Expected results:</b>
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<li>Slides treated with GOPTS should show fluorescence.</li>
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<li>Untreated cells should show no fluorescence.</li>
  
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will take many hours in a fridge, over an hour on ice, the fastest is to use an
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</ul>
  
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ice/water slurry.
 
  
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<br>1. Take a single colony from an LB agar plate, inoculate in 5 ml of LB broth
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</p>  
  
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and incubate for overnight, 200 rpm at 370C.
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<ul>
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<p>________________________________________________________________________________________________________________________________________________</p>
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<h3> Experiment 2: Testing the expression of zinc finger proteins on the surface of E. coli cells upon induction with IPTG </h3>
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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.
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<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>
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<br><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>
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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>
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<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>
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<b>Expected results:</b>
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<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>
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<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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</ul>
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</p>
  
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<br>2. Dilute 200 µL of culture in 100 ml of LB broth and incubate with shaking
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<ul>
  
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until the culture reaches an OD value of 0.30-0.35. You can make
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<p>________________________________________________________________________________________________________________________________________________</p>
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<p>
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<H3> Experiment 3: Reciprocal experiment - binding of fluorescently labelled oligonucleotides to immobilised cells </H3>
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<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>
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Objective: To ensure that our zinc finger proteins are able to bind to fluorescently labelled oligonucleotides which contain the zinc finger binding domain.
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<br><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>
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<li>Fluorescently labelled oligonucleotides (containing the zinc finger binding domains) were added.</li>
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<li>Binding of the zinc finger proteins to the fluorescent oligonucleotides allows visualisation of the cells by immunofluorescence microscopy. </li>
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<b>Expected results:</b>
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<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>
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<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>
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<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>
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<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>
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</p>
  
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larger volumes if you like.
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</ul>
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<br>
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<br>
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<br>
  
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<br>3. Keep the culture on in ice/water bath for 30 min, mixing every now and
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<p>________________________________________________________________________________________________________________________________________________</p>
  
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again to ensure even cooling. Make sure the culture never warms
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<b><H3> Future Experiments </H3></b>
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<p>Due to time restrictions, we were unable to carry out the following experiment. This experiment involved ‘painting a microscopic picture’ with our fluorescent cells. This was to demonstrate specific localisation of cells controlled by our programmable Brixells.
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</p><br>
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<ul>
  
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above 4ºC from this point on.  
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<p>
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<H3> Experiment 4: Binding fluorescent zinc finger expressing cells to oligos on a glass slide </H3>
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Objective: To see whether we can control the precise locations of different coloured cells through binding of the zinc finger proteins to oligonucleotides in specific positions on a glass slide.
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<br><br><li>A range of zinc finger expressing cells would be prepared (with the cells for each zinc finger corresponding to one particular colour).</li>
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<li>Different oligos would be placed on a glass slide in specific places (either by hand, or using a parafilm template). The positioning of these oligos would determine where cells of any given colour are able to bind.
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</li>
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<li>Using immunofluorescence microscopy, we would visualise our slide, and see cells of different colours coming together to form a ‘picture’.
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<img src="https://static.igem.org/mediawiki/2015/0/00/Warwick_Diagram_of_specific_zinc_finger_DNA_binding.jpeg" border="50px">
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</li>
  
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<br>4.Transfer to pre-cooled centrifuge tubes and centrifuge at 3000 rpm at 40C
 
  
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for 10 min.
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</ul>
  
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<br>5. Remove the supernatant, then resuspend the bacteria in 10 ml of 100 mM
 
  
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CaCl2, mix well and incubate for 30 min on ice.
 
  
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<br>6. Centrifuge at 3000 rpm at 40C for 10 min. Discard the supernatant, then
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<p>________________________________________________________________________________________________________________________________________________</p>
  
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add 10 ml of 100 mM MgCl2, mix then incubate for 30 min on ice.
 
  
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<br>7. Centrifuge at 3000 rpm at 40C for 10 min and add 2 ml CaCl2 with 10%
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<ul>
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<p>
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<H3> Experiment 5: Binding fluorescent zinc finger expressing cells to oligonucleotide adhesive </H3>
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Objective: To see whether we can control the binding of different cell types to an engineered DNA structure.
  
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glycerol, resuspend bacteria.  
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<br><br><li>Make more DNA origami Y structures.</li>
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<li>Grow up three different types of zinc-finger expressing cells.
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</li>
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<li>Wash the cells and origami together, and allow to sit for 2-3 hours.
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</li>
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<li>Using immunofluorescence microscopy, we would visualise our slide, and see cells of different colours coming together to form a ‘picture’.
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</li>
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<img style="-webkit-user-select: none" src="https://static.igem.org/mediawiki/2015/1/14/Warwick_Diagram_of_specific_zinc_finger_DNA_binding_2.jpeg">
  
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<br>8. Transfer 50 µL of culture into pre-cooled 1.5mL eppendorf tubes (on ice,
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</ul>
  
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or do this in the cold room). Snap freeze cells in liquid nitrogen and
 
  
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transfer to -80 freezer.
 
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</p>
 
  
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<h3> Chemical transformation </h3>
 
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<h5> Puts your plasmid into cells </h5>
 
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<p> 1. Take chemically competent cells (-80C freezer) and thaw on ice/water mix.
 
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<br> 2. Add plasmid DNA to 50uL of competent cells: for minipreps 0.5-1 uL of DNA is enough, for ligations, use 5-10 uL. Mix thoroughly.
 
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<br> 3. Leave on ice for 30-45 minutes. Turn on the waterbath set to 42C, so it reaches the target temperature over this time.
 
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<br> 4. Heat shock the cells at 42C for 30 seconds. This will create pores in the competent cells through which the plasmid can enter into the cell.
 
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<br> 5. Put back on ice for 2 minutes. This allows the cell to recover and begin repairing the pores, preventing cell death.
 
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<br> 6. Add 950uL of growth media (e.g. SOC, LB, etc.) bringing the volume up to ~1000uL
 
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<br> 7. Incubate with shaking at 37C for 45-60 minutes.
 
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<br> 8. Plate 200uL on appropriate antibiotic: If using a ligation (or anything likely to have low efficiency) centrifuge the cells first at 8000rpm for 2 minutes and resuspend in 200uL of media then plate everything. If there are still some cells left after plating, the rest can be kept up to 3 weeks in a 4 degree fridge.
 
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<br> 9. Incubate overnight at 37C.
 
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</p>
 
  
  
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<p>
 
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<H3> Ethanol Precipitation Protocol </H3>
 
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<H5>Simple method for gel extraction</h5>
 
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<p>Ethanol Precipitation of DNA Reagents Needed:</p>
 
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<br>• 3 M sodium acetate pH 5.2 or 5 M ammonium acetate
 
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<br>• DNA
 
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<br>• 100% ethanol
 
  
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<h5>Extracting gel</h5>
 
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<p>After running a gel and identifying the band that contains the DNA that you wish to extract, simply use a scalpel to cut around the band leaving as little excess agarose gel as possible.</p>
 
  
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<H5>Protocol</H5>
 
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<br>1. Measure the volume of the DNA sample.
 
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<br>2. Add 1/10 volume of sodium acetate, pH 5.2, (final concentration of 0.3 M) - These amounts assume that the DNA is in TE only; if DNA is in a solution containing salt, adjust salt accordingly to achieve the correct final concentration.
 
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<br>3. Mix well.
 
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<br>4. Add 2 to 2.5 volumes of cold 100% ethanol (calculated after salt addition).
 
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<br>5. Mix well.
 
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<br>6. Place on ice or at -20 degrees C for >20 minutes.
 
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<br>7. Spin a maximum speed in a microfuge 10-15 min.
 
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<br>8. Carefully decant supernatant.
 
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<br>9. Add 1 ml 70% ethanol. Mix. Spin briefly. Carefully decant supernatant.
 
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<br>10. Air dry or briefly vacuum dry pellet.
 
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<br>11. Resuspend pellet in the appropriate volume of TE or water.
 
  
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</p>
 
  
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<h3> Double restriction digestion for NEB restriction enzymes </h3>
 
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<h5> Cuts a select piece of DNA from a plasmid </h5>
 
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<h5> Protocol </h5>
 
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<p> Set up the reaction as follows:
 
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<br> - 1ug DNA
 
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<br> - 5uL 10x digest buffer (use NEB cloner to find which buffer works best with which enzyme)
 
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<br> - 1uL or 10 units of first enzyme
 
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<br> - 1uL or 10 units of second enzyme
 
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<br> - Up to 50uL nuclease-free water
 
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<br>
 
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<br> Incubate at 37C for 1 hour. If the enzymes being used are both time save qualified, this can be reduced to 5-15 minutes, but incubating for longer is still recommended.
 
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<br> Add the reagents into the mix from largest volume to smallest, always finishing with adding the enzymes in last.
 
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<br> If multiple restriction digests are being set up, a master mix containing everything but the sample DNA can be made with the condition that the concentrations of the different sample DNA are similar or equal.
 
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</p>
 
  
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<h3> Bacterial immunofluorescence protocol </h3>
 
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<h5> For preparing slides to be visualised under fluorescent microscopy </h5>
 
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<p> 1. For every cell type that needs testing, grow a culture of bacterial cells in 5mL LB
 
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(+antibiotics) overnight at 37 ˚C.
 
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<br>2. Next morning, take OD600 of the cultures (OD of 1 for E. coli corresponds to ~10^8 cells/mL),
 
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and dilute into 2 fresh 5mL LB tubes (+antibiotics) to OD600 of ~0.01. To one of these tubes,
 
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add IPTG to end concentration of 1mM. Incubate both tubes in a 37 ˚C shaker.
 
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<br>3. After ~3 hr of incubation, start monitoring OD of the cultures every half hour. We want to fix
 
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these cells at an OD600 of ~0.5.
 
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<br>4. As soon as a culture reaches OD600 of ~0.4-0.5, spin down 1mL of the culture in an Eppendorf
 
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tube at 8000xg (=rcf) for 1 min, and carefully discard the supernatant (be careful so as to
 
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only remove the supernatant, without disturbing the cells in the pellet).
 
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<br>5. Re-suspend the pellet in 1mL 1xPBS by pipetting up and down 5 times. Spin down the cells at
 
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8000xg (=rcf) for 1 min, and carefully discard the supernatant.
 
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<br>6. Repeat the PBS wash in Step-5 two more times, but this time only use 0.5mL PBS.
 
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<br>7. Now, re-suspend the cells in 0.5mL 1xPBS by pipetting.
 
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<br>8. Mix 500 uL Blocking buffer with the annealed oligo (5.13uL) for each cell type in a separate
 
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Eppendorf tube, then add this to your cells.
 
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<br>9. Spin down the cells at 8000xg (=rcf) for 1 min, and carefully discard the supernatant.
 
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<br>10. Do 1x PBS wash (0.5mL PBS).
 
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<br>11. Now, fix the cells (in the tube itself) by resuspending in 1xPBS+4%(para)formaldehyde (we used glutaraldehyde but it fulfills the same purpose) (500uL). Incubate at room temperature for 20 min.
 
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<br>12. Do 1x PBS washes (0.5mL PBS)
 
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<br>13. Drop 50uL of the resuspension on a coverslip (round coverslips preferred), and incubate at
 
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37 ˚C until it is completely dry. Once dry, save the coverslip at RT until all the cultures have
 
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been processed similarly.
 
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<br>14. Add a drop of the mounting medium (ProLong Diamond Antifade Reagent, Fisher
 
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#15372192) on a glass slide and place the coverslip on top of it (bacterial-side-down).
 
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<br>15. Seal the edges of the cover-slip with nail-polish, and save in the fridge (4˚C), for later
 
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visualization.
 
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</p>
 
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<br>
 
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<br>
 
  
  

Latest revision as of 18:15, 18 September 2015

Warwick iGEM 2015

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