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.
Design by Warwick iGEM 2015