Difference between revisions of "IGEM:Harvard/2006/Final Presentation"
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Latest revision as of 23:55, 3 November 2006
Cell Surface targeting (connects from nano)
Streptavidin is a protein which has been found to bind very strongly to biotin, hence its laboratory use in binding biotinylated DNA and peptides. The big idea here is that the expression of streptavidin on the surface of a cell would make up an artificial targeting system for any biotinylated peptide or DNA, such as a DNA nanostructure.
We used the Lpp-OmpA surface display vehicle, published by the Georgiou. The vehicle is a fusion of the signal peptide for Lpp, lipoprotein, and the transmembrane domains of OmpA, outer membrane protein A (they used both 1 and 5 domains). They successfully expressed proteins like beta-lactamase or antibody chains. We would be displaying streptavidin.
So how do we put the protein domains together? Standard BioBrick parts include a spacer nucleotide between the part and the flanking XbaI and SpeI restriction sites. It was researched at the Silver lab that by leaving out these spacer nucleotides, you can maintain the reading frame between protein domains and thus splice domain coding sequences together for a fused protein. And so the full construct would contain a promoter, a ribosome binding site, and a fused protein with the Lpp signal peptide, OmpA with 1 or 5 transmembrane domains, and streptavidin. We had a monomeric form from the Ting lab, and a single-chain dimer from the Aslan lab.
In a western blot probing for the histidine tags at the end of these proteins, we found bands at the expected sizes, and a parallel probing for streptavidin showed the same bands. Future plans include testing the functionality of these constructs by probing with a biotinylated, fluorescently tagged oligonucleotide.
1. Nanostructures including a sentence about targeting (8 minutes).
2. Cell surface targeting (4 minutes).
3. Cyanobacteria (8 minutes).