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Cell Surface Targeting

Cell Surface Targeting. 8/7/06. Progress/agenda. Streptavidin BioBricks Sequencing for QuikChange-mutagenized single-chain dimer streptavidin showed 141bp deletion #394-534

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Cell Surface Targeting

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  1. Cell Surface Targeting 8/7/06

  2. Progress/agenda • Streptavidin BioBricks • Sequencing for QuikChange-mutagenized single-chain dimer streptavidin showed 141bp deletion #394-534 • Performed QuikChange mutagenesis on SCD streptavidin clones originally received in pET22 vector. Received new streptavidin clone (pTSA13) from Dr. Takeshi Sano • Plan to extract mutagenized clones and pTSA13 by PCR • Lpp-OmpA assembly with streptavidin • Ligations have been unsuccessful, possibly due to attempted use of NEB T4 DNA Ligase when the Roche Rapid DNA Ligase ran out. Also received two Lpp-OmpA constructs from Georgiou lab. • Plans to retry ligations with new Rapid DNA ligase kit, and BioBrick-PCR Georgiou constructs • B0032 (ribosome binding site BioBrick) • Transformed and confirmed DNA sequence • Plans to ligate R0010 (Lac promoter) and B0032 upstream of Lpp-OmpA-Strep

  3. Adaptamers

  4. Outline • Gel shifts • Streptavidin beads • New designs • What’s next

  5. Outline • Gel shifts • Streptavidin beads • New designs • What’s next

  6. T20 A20 S20 T35 A35 S35 T50 Link-aptamer A50 S50

  7. Shift! (but is it due to thrombin?) 1 2 3 4 5 6 7 8 9 10 11 12 • 1: SeeBlue Plus 2 ladder • 2: thrombin • 3: thrombin + T35 • 4: thrombin + S35 • 5: thrombin + T35 + S35 • 6: 1kb+ ladder • 7: T35 • 8: T35 + thrombin • 9: S35 • 10: S35 + thrombin • 11: S35 + T35 • 12: S35 + T35 + thrombin 188 98 62 49 38 12% polyacrylamide gel; Tris-Glycine Buffer White background stained for protein; black background stained for DNA

  8. Hopefully. 1 2 3 4 5 6 7 8 9 10 11 12 • 1: SeeBlue Plus 2 ladder • 2: thrombin • 3: thrombin + T35 • 4: thrombin + S35 • 5: thrombin + T35 + S35 • 6: 25 bp ladder • 7: T35 • 8: T35 + thrombin • 9: S35 • 10: S35 + thrombin • 11: S35 + T35 • 12: S35 + T35 + thrombin 188 98 62 49 38 100 75 50 25 6% polyacrylamide gel; .5X TBE Buffer Meanwhile, pure thrombin aptamer does not show a shift. Larger weight of A35 (=S35+T35) may be factor.

  9. No shift for A20 1: SeeBlue Plus 2 ladder 2: thrombin 3: thrombin + T50 4: thrombin + S50 5: thrombin + T50 + S50 6: 25bp ladder 7: T50 8: T50 + thrombin 9: S50 10: S50 + thrombin 11: S50 + T50 12: S50 + T50 + thrombin 188 98 62 49 38 100 75 50 25 12% polyacrylamide gel; Tris-Glycine Buffer

  10. Shift for A50 1 2 3 4 5 6 7 8 9 10 11 12 1: SeeBlue Plus 2 ladder 2: thrombin 3: thrombin + T50 4: thrombin + S50 5: thrombin + T50 + S50 6: 1kb+ ladder 7: T50 8: T50 + thrombin 9: S50 10: S50 + thrombin 11: S50 + T50 12: S50 + T50 + thrombin 188 98 62 49 38 200 100 12% polyacrylamide gel; Tris-Glycine Buffer

  11. No shifts for streptavidin (A20, A35) 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12 • 1: SeeBlue Plus 2 ladder • 2: streptavdin • 3: streptavidin + TN • 4: streptavidin + SN • 5: streptavidin + TN + SN • 6: ladder N= 20; 12% gel, Tris-glycine buffer N= 35; 6% gel, .5X TBE 7: TN 8: TN + streptavidin 9: SN 10: SN + streptavidin 11: SN + TN 12: SN + TN + streptavidin

  12. Outline • Gel shifts • Streptavidin beads • New designs • What’s next

  13. Streptavidin Beads • First try: botched control, botched washes

  14. S0: streptavidin aptamer (40 bp single stranded) SB: streptavidin beads TB: thrombin beads Second try 1 2 3 4 5 6 7 8 9 1: ladder 2: 40 pmol S0 3: 10 uL SB + 40 pmol S0 4: 25 uL SB + 40 pmol S0 5: 50 uL SB + 40 pmol S0 6: 100 uL SB + 40 pmol S0 7: 150 uL SB + 40 pmol S0 8: T35 (50bp, single stranded oligo) 9: 150uL TB + 40 pmol S0 60 50 40 30 20 10 Above: Elutions with streptavidin Below: Washes with buffer 60 50 40 30 20 10

  15. Outline • Gel shifts • Streptavidin beads • New designs • What’s next

  16. Designing adaptamer linkers • Problems: adaptamer linker sequences can basepair to aptamer regions or to self

  17. Designing adaptamer linkers • Program to evolve optimal adaptamers • Minimize number of 4-mers that have bad basepairing properties • Metropolis algorithm • Start with random sequence, repeatedly identify “worst” 4-mer, mutate with some probability

  18. Results Thrombin aptamer Streptavidin aptamer T50 S50

  19. New designs Streptavidin aptamer Thrombin aptamer

  20. Outline • Gel shifts • Streptavidin beads • New designs • What’s next

  21. What’s next • Ordered fluorescently labeled aptamers; will try nitrocellulose filter assay • Order new designs with/without fluorophores. • Retry streptavidin-agarose bead assay with full adaptamers; maybe even see if thrombin can bind together • FRET experiment? • Design Biacore experiment.

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