Experiments
What follows is the general step by step guide of how we set about creating, testing and refining our idea.
Experiment 1: Establishing Anchor Protein
1. Take a briobrick part (JO4450) and transform it into a chemically competent cell (Top 10)
2. Grow cells overnight then miniprep them to extract the transformed plasmid
3. Double digestion of plasmid to cut out the RFP gene, leaving only the plasmid backbone
4. PCR the full construct G-Block (containing Zif268 and Lpp_OmpA) and digest the product with the corresponding enzymes
5.
Golden gate assembly of plasmid backbones
and full construct.
6. Transform electrocompetent E.
coli cells with full plasmid
7. Grow cells
8. Miniprep full construct plasmid from modified cells
9. Digest plasmid with enzymes matching the cut sites around the Lpp_OmpA gene
10. PCR the rest of the anchor proteins from the G-blocks ordered (INP, PGSA, and BCLA)
11. Ligate the anchor proteins into digested full construct plasmid
12. Transform all plasmids into electrocompetent E.
coli - end product: four different types of cell, each expressing the full construct with slightly different transmembrane domains
13. Prepare transformed cells for microscopy (mount cells on glass slides)
14. Bind antibodies to FLAG tag - measure under fluorescent microscope
15. Determine whether the gene is being expressed and whether the expressed protein is being transported and is binding to the membrane of the E.
coli
16. Compare intensities - select anchor protein that fluoresces brightest, this anchor protein with be used for all subsequent experiments.
Experiment 2: Establishing Zinc Finger DNA Binding Proteins
1. Miniprep full construct plasmid from the first experiment
2. Digest plasmid with enzymes corresponding to the cut sites around the Zif268 gene
3. PCR zinc finger genes from the G-blocks ordered
4. Golden gate assembly of digested full construct plasmid and the three zinc finger PCR products
5. Transform electrocompetent E.
coli with each modified plasmid - end product: four different types of cells, each with the same anchor protein but different zinc finger DNA binding domains
6. Prepare the cells for microscopy by binding them to slides
7. Prepare four different sets of fluorescent oligos that each contain the target sequence for a zinc finger
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
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
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
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