Difference between revisions of "Team:Warwick/Protocols"

Line 30: Line 30:
 
<br>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.
 
<br>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.
 
<br>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.
 
<br>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.
 +
<br>Expected results:
 +
<br>Slides treated with GOPTS should show fluorescence.
 +
<br>Untreated cells should show no fluorescence.
 +
<br>This is a control to ensure that our zinc finger binding domains can be bound to glass slides.
  
  
Line 35: Line 39:
 
</p>  
 
</p>  
  
−
<h3> Chemical transformation </h3>
+
<h3> Experiment 2: Testing the expression of zinc finger proteins (on the surface of E. coli cells) upon induction with IPTG </h3>
−
<h5> Puts your plasmid into cells </h5>
+
<br>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.
−
<p> 1. Take chemically competent cells (-80C freezer) and thaw on ice/water mix.
+
<br>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.
−
<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.
+
<br>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. (put link to Warwick iGEM 2015 bacterial immunofluorescence protocol).
−
<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.
+
<br>Expected results:
−
<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.
+
<br>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.
−
<br> 5. Put back on ice for 2 minutes. This allows the cell to recover and begin repairing the pores, preventing cell death.
+
<br>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.
−
<br> 6. Add 950uL of growth media (e.g. SOC, LB, etc.) bringing the volume up to ~1000uL
+
 
−
<br> 7. Incubate with shaking at 37C for 45-60 minutes.
+
−
<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.
+
−
<br> 9. Incubate overnight at 37C.
+
 
</p>
 
</p>
  
  
 
<p>
 
<p>
−
<H3> Ethanol Precipitation Protocol </H3>
+
<H3> Experiment 3: Reciprocal experiment - binding of fluorescently labelled oligonucleotides to immobilised cells </H3>
−
<H5>Simple method for gel extraction</h5>
+
<br>E. coli cells expressing each of our 4 zinc finger proteins were immobilised onto glass slides (put link to bacterial immunofluorescence protocol updated).
−
<p>Ethanol Precipitation of DNA Reagents Needed:</p>  
+
<br>Fluorescently labelled oligonucleotides (containing the zinc finger binding domains) were added.
−
<br>• 3 M sodium acetate pH 5.2 or 5 M ammonium acetate
+
<br>Binding of the zinc finger proteins to the fluorescent oligonucleotides allows visualisation of the cells by immunofluorescence microscopy. 
−
<br>• DNA
+
<br>Expected results:
−
<br>• 100% ethanol
+
<br>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.
 +
<br>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.
 +
<br>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.
 +
<br>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.
 +
</p>
 +
 
  
 
<h5>Extracting gel</h5>
 
<h5>Extracting gel</h5>

Revision as of 14:04, 17 September 2015

Warwick iGEM 2015

Open Menu