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

2. 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.

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

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

5. 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

6. 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)

7. 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)

8. 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