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Harvard iGEM 2006

Harvard iGEM 2006. Our Vision: modular drug delivery. 1. 2. cell A. cell B. cell C. targeting. packaging. Why DNA ?. Strong Watson-Crick base pairing Covalent modifications Relatively inexpensive Self-assembles Highly programmable. How: engineered crossing over.

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Harvard iGEM 2006

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  1. Harvard iGEM 2006

  2. Our Vision:modular drug delivery 1. 2. cell A cell B cell C targeting packaging

  3. Why DNA? • Strong Watson-Crick base pairing • Covalent modifications • Relatively inexpensive • Self-assembles • Highly programmable

  4. How:engineered crossing over

  5. How:scaffolded origami N. Seeman P. Rothemund W. Shih et. al

  6. Our Design:a novel structure courtesy Shawn Douglas

  7. Our Design:a novel structure courtesy Shawn Douglas

  8. Evidence:EM images 15.5 nm

  9. Evidence:EM images 27.6 nm 30.5 nm

  10. Evidence:protection assay Protected biotin on the inside of our container from large streptavidin beads Streptavidin bead Streptavidin bead Payload protected Control: biotin bound

  11. Harvard iGEM 2006

  12. Adaptamers Thrombin aptamer Streptavidin aptamer Linking region Thrombin Streptavidin Aptamer: nucleic acid sequence that can bind a substrate with high specificity and affinity.

  13. 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.

  14. Adaptamer testing streptavidin adaptamer Adaptamer + thrombin thrombin anti-thrombin antibody (-) control: naked beads Bead surface

  15. Adaptamer quenching Displacement strand + +

  16. Streptavidin on the cell surface Streptavidin • 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

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

  18. Standard BioBrick 5’…TCTAGAG XbaI TACTAGT…3’ SpeI Protein domain BioBrick 5’…TCTAGA_ XbaI _ACTAGT…3’ SpeI 6-bp mixed site Reading frame maintained TCTAGT Mixed Lac Promoter RBS Lpp OmpA Streptavidin BioBricks assemblyof protein domains I. Phillips P. Silver 2006

  19. Anti-Histidine Anti-Streptavidin Expression of fusion protein in frame • Inducible expression • Histidine tag at end, so construct in frame • Streptavidin domain recognized by Ab

  20. Harvard iGEM 2006

  21. From cyanobacteria... • Photosynthetic • Circadian rhythm • Evolved over billions of years ...to E. coli • Model organism for synthetic biology • BioBrick registry

  22. Applications of a Bio-oscillator 12PM 12PM • Clock • Nightlight • Timed drug delivery • Pharmaceutical processes • Bio-circuitry • Investigate natural systems A+E->F A+B->C 6PM 6AM 6PM output http://www.cellzome.com/img/pathways.jpg D+C->E D+F->P 12AM 12AM Time

  23. The Repressilator Cyanobacteria Bio-oscillator Lac λ-cI Tet Elowitz et al. 2000 ` Fluorescence Time 10h 24h

  24. The Kai Clock in Cyanobacteria KaiCautophosphorylates and dephosphorylates KaiA promotes phosphorylation KaiB inhibits KaiA Transcription-translation independent Period: 14-60 h A B C P P P P P P b Phosphorylation of KaiC output Time

  25. Achievements Goal: reconstitute the cyanobacteria Kai oscillator in E. coli Created KaiA, KaiB, and KaiC BioBricks. Combined the above with registry parts to form functional BioBricks. Expressed Kai proteins in E. coli and verified interaction.

  26. Results: Constructs Created We’ve made the following constructs: Kai genes Lac promoter + Kai genes KaiA + KaiC and KaiB + KaiC (with promoters)

  27. Results: Proteins Interact in E. coli Constructs transformed in E. coli Cultures sampled at OD 0.5 Western blot probed with anti-KaiC antibodies Western blot image

  28. Results: Proteins Interact in E. coli Western blot image A A C B C C

  29. Results: Proteins Interact in E. coli Western blot image A A C B C C

  30. Results: Proteins Interact in E. coli Western blot image A A C B C C

  31. Results: Proteins Interact in E. coli Western blot image Conclusion: • KaiA and KaiC are expressed and interacting • KaiB not verified, but results consistent with predictions

  32. Further challenges A B A A A B B B C C C C A Time B C Verify oscillation in E. coli. Synchronization problem • Between cells • Within cell Solution: pulsed expression P P P P P P

  33. Harvard iGEM 2006

  34. Acknowledgements Advisors George Church Pamela Silver William Shih Radhika Nagpal Jagesh Shah Alain Viel Teaching Fellows Nicholas Stroustrup Shawn Douglas Chris Doucette

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