Team:Warwick/Microscopy
Fluorescent Microscopy
Qualitative Proof of Concept
In order to visualise our cells and determine whether the modified zinc finger protein was binding to the surface of our E. coli cell, we also included a FLAG tag domain in the protein.
This allowed us to use anti-FLAG antibodies to conduct an immuno-fluorescence test to see the location of the protein in the cells. The immuno-fluorescence test is conducted by fixing our modified cells to slides and washing over them with first a primary anti-FLAG antibody, a polypeptide which recognises and binds the FLAG-tag domain we added to our protein, then a secondary antibody that binds to the primary. This secondary antibody is fluorescent. Under a fluorescent microscope this fluorescence should be visible, and it's presence is how we determined qualitatively that the zinc fingers were binding to the surface of the cell. The protocol for this can be found here.
Reciprocal Test
After determining that our modified zinc fingers were being expressed on the surface of the E. coli cell, we set out to demonstrate whether our modified cell could bind DNA discriminately. If the cell binds DNA, but any old piece of DNA binds instead of the DNA we have selected, this would go against the aims of our project.
We set out to demonstrate this using fluorescein bound oligonucleotides that contained our zinc finger target DNA sequence. By fixing different cells to slides and washing them with different oligos we could determine whether the zinc fingers bind only target DNA, or any DNA. This test could be conducted quantitatively by doing a photon count using the fluorescent microscope. This can then be used to determine which of the four zinc fingers we tested bound the most oligo, and therefore had the strongest binding affinity with its target sequence. This information could potentially be important for any further research conducted in this field.
