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Encapsulation for Drug Delivery

Encapsulation for Drug Delivery. 7 th August iGEM Team 2009. Kun Nuri Royah Tianyi. Charles Dave Dineka James. The Project. “Auto-encapsulation of protein drugs for release in the small intestine.”. Gantt Chart. Modules 1 & 2: Update. M1 - Hunt for Cellulase.

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Encapsulation for Drug Delivery

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  1. Encapsulation for Drug Delivery 7th August iGEM Team 2009 Kun Nuri Royah Tianyi Charles Dave Dineka James

  2. The Project “Auto-encapsulation of protein drugs for release in the small intestine.”

  3. Gantt Chart

  4. Modules 1 & 2:Update

  5. M1 - Hunt for Cellulase • No clone readily available. Professor input?

  6. M2 - Sodium Acetate • Food grade • RAPID inducible crystallisation • Exothermic • Dissolves under acidic • conditions

  7. The TimerModule Integration

  8. TimerDesign Rules 1. Try relyonactivation, notrepression. (Problemwithdegradationrates.) 2. Inputs mustaccumulateover time toseedelays. 3. Lastpromotermustbe as “non-leaky” as possible. 4. Tunability - linkedto inducible promotor.

  9. Timer - Logic A PoPS (Encapsulation) AB B Inducible Constitutive

  10. Kirsten’s favourite Timer – v1 Tunability PBAD PLux LuxI M2 Lux R + I PoPS (Encapsulation) X LuxR Start

  11. PAH or Cellulase M1 ! • Conflict between: • Start of experiment • Threshold setting X PBAD LuxR LuxI Timer M2 PluxR Encapsulation (M2) genes

  12. PAH or Cellulase M1 ! • Conflict between: • Start of experiment • Threshold setting PBAD J23114 LuxR LuxI Timer PluxR Encapsulation (M2) genes M2

  13. Timer – v2 X Bba_K145013 Anti-LuxI PBAD LuxI PLux M2 Lux R + I PoPS (Encapsulation) J23114 LuxR

  14. Tunability of IPTG in lacY-strains Induction of lac-regulated promoter in lacY- and lacY+ strains, from Jensen et al. Eur.J.Biochem, 211, 181-191 (1993)

  15. PluxR Output

  16. Timer – v4 Td (lactonase) =4.7hrs PTET Lactonase Bba_C0060 PLAC luxI tetR Ts (TetR) PLux M2 Ts (LuxI) Lux R + I PoPS (Encapsulation) J23114 LuxR

  17. Dry LabPreparation

  18. Dry Lab • So far… • Designed & ordered primers • Designed TaqI, DpnII & cI BBs • Testing construct design • Clones ordered (Kirsten) • Started step-by-step protocols & shopping list • Next week… • Place MrGene order • SLIC preparation • Complete step-by-step protocols & shopping list

  19. BioBricks Remaining… M3 BBa_K098995 [Harvard ‘08]

  20. Wet LabParts, Testing & Progress

  21. So Far… • Competent cells – 4 x 10^6 cells/ug DNA • 0 contaminants • No native resistance

  22. So Far… • Registry - 8 taken out

  23. Next week… • Registry – extract timer BBs • Start PCR – M1 (2), M2 (5) & M3 (1) • Start to characterise promoters (Jason Kelly) • Organise teams (1 bioscientist, 1 bioengineer) • - team 1 = PCR • - team 2 = promoter testing

  24. Summary - Checklist Weekly 5 Aims: • Trade name • Assay Protocols • Cloning Strategies • Finalise genetic circuit • Order BioBricks • Primer design • Wet lab plan

  25. Summary - Checklist Week 6 Aims: • Place MrGene Order (Wednesday) • Assay Protocols (Monday) • Cloning Strategies (Monday) • Wet lab – PCR & test promoters & extract BBs • Dry lab tutorial

  26. Any questions?

  27. Timer – v3 (Lock/Key system) PtetR Bba_K145300 J23109 Lactonase LVA BBa_K145102 Lock + Key PLux J23114 LuxI M2 Lux R + I PoPS (Encapsulation) J23114 LuxR

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