Adaptamers Thrombin-binding domain Streptavidin-binding domain

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Presentation transcript:

Adaptamers Thrombin-binding domain Streptavidin-binding domain Linking region Thrombin Streptavidin

Why develop adaptamers? To offer a new way to target substrates to cells. To create a tool for studying cell-cell interactions. To provide a foundation for engineered catalysts. happy colorful things.

Adaptamer testing Bead surface Adaptamer = A15 Control: no thrombin streptavidin adaptamer Adaptamer = A15 thrombin anti-thrombin antibody Control: no thrombin Bead surface

Adaptamer quenching… + + Results: Displacement strand change color of displacement strand, stop blinding pp (yellow) No displacement strand added Displacement strand added Background fluorescence +

Streptavidin on the cell surface Binds strongly to biotin molecule Used to bind biotinylated nucleic acids or peptides Big idea: Streptavidin protein expressed on the cell surface can be used to target any biotinylated DNA/protein to the cell surface. McDevitt, 1999

Lpp-OmpA surface display J. Francisco, C. Earhart, G. Georgiou, 1992 periplasm extracellular outer membrane Surface protein Lpp (lipoprotein) signal peptide OmpA (outer membrane protein A) transmembrane domains (1 or 5)

BioBricks assembly of protein domains I. Phillips P. Silver 2006 5’…TCTAGAG XbaI TACTAGT…3’ SpeI Standard BioBrick 5’…TCTAGA_ XbaI _ACTAGT…3’ SpeI Protein domain BioBrick TCTAGT Mixed 6-bp mixed site Reading frame maintained Promoter RBS Lpp OmpA Streptavidin O1 SM (Ting) O5 SD (Aslan)

Expression of construct Anti-His L-O1-SM L-O5-SD L-O1-SD L-O5-SM L-O1-SM Anti-Streptavidin L-O5-SD L-O1-SD L-O5-SM