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The Universal Pill

The Universal Pill. IGEM Presentation 17 th July 09 James Field Dineka Khurmi Nuri Purswani Kun Xue. Project Description. Problem for oral delivery of peptides 1 pill = 1 drug High manufacturing cost Variable peptide half life Solution… User defined drug production….

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The Universal Pill

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  1. The Universal Pill IGEM Presentation 17th July 09 James Field Dineka Khurmi Nuri Purswani Kun Xue

  2. Project Description • Problem for oral delivery of peptides • 1 pill = 1 drug • High manufacturing cost • Variable peptide half life • Solution… • User defined drug production… Specification Design Modelling Implementation Testing/Validation

  3. The Universal Pill • Multiple inputs enable drug selection • Offers uniformity • Direct packaging • Fresh peptide production • Dosage control • Reduced loss of peptide Capsule Bacteria Specification Design Modelling Implementation Testing/Validation

  4. Current Methods Specification Design Modelling Implementation Testing/Validation

  5. Integrated Solution Specification Design Modelling Implementation Testing/Validation

  6. Chosen Solution • Polysaccharide encapsulation • of chassis • Combining polysaccharide & symbiotic microbe delivery offers the following advantages: • Synthesis on demand without risk of GMO • Protein is not denatured during storage & transport Capsule Bacteria Specification Design Modelling Implementation Testing/Validation

  7. Mechanism Overview • Polysaccharide encapsulation of chassis. • Peptide synthesis prior to consumption. Encapsulate Express Kill Release Specification Design Modelling Implementation Testing/Validation

  8. Light Trigger Logic Circuit 0 R = 0 B = 0 No production Drug 1 B R = 0 B = 1 Drug 1 R Drug 2 0 R = 1 B = 0 Drug 2 R = 1 B = 1 Drug 3 Drug 3 Specification Design Modelling Implementation Testing/Validation

  9. Proposed Applications

  10. Chassis Criteria • Non pathogenic strain • Large Biobrick availability • Expertise in college • Freeze dry Testing/Validation

  11. Chassis considerations

  12. Capsule Design Overview • Encapsulation • Storage • Protein expression • 4) Acid resistance • 5) Release

  13. 1) Encapsulation • enhances cell resistance to freezing and freeze-drying (for storage) • added convenience and reduced packaging costs • longer stability and viability during storage

  14. Encapsulation Method Comparison

  15. Encapsulation details An in situ method for cultivating microorganisms using a double encapsulation technique Eitan Ben-Dov1,2, Esti Kramarsky-Winter3,4 & Ariel Kushmaro1,5

  16. 2) Storage • Short term storage-up to a month • Nutrient agar • Keep in a sealable container • Storage in refrigerator • Long term storage • Inclusion of glycerol • storage in freeze-dried form • freeze at -20°C or -80°C

  17. Freeze drying

  18. 3) Protein expression • Transparency to light important for light inputs reaching cells • Alginate is transparent • Transparency type nutrient agar

  19. Protein Deposition • Proteins expressed are exported from the cell into the nutrient agar • Proteins stored in pores of nutrient agar until release

  20. 4) Resistance to stomach acidity • Exposure of bifidobacteria to simulated gastric juice at pH 2.0 • Diameters of 40–80 μm - insignificant protection • 1–3 mm - microspheres protected entrapped cells Encapsulation in alginate-coated gelatin microspheres improves survival of the probiotic Bifidobacterium adolescentis 15703T during exposure to simulated gastro-intestinal conditions N.T. Annana, A.D. Borzaa and L. Truelstrup Hansen

  21. Resistance to Acidity • When pH is lowered below the pKa values of d-mannuronic and l-guluronic acid (3.6 and 3.7, respectively), alginate is converted to alginic acid with release of calcium ions • Stomach pH is at 1-3 • Disintegration times for alginate-coating was 120 min

  22. 5) Release • Full degradation of alginate coat in intestines • Protein in nutrient agar now released

  23. The Vision LOAD PILL SELECT DRUG SELECT DOSE COMPETE

  24. Black Box Light Chemical

  25. BLACK BOX: Modules Drug Control Dose Control Light Sensing Frequency Wavelength Timer Peptide synthesis Restriction enzyme synthesis

  26. INPUT: Encoding with Light Wavelength: Pulse: Cph8 1 0 YcgF/YcgE Time Drug Choice Dosage

  27. Wavelength Encoding Input A B C Output 1, 0 1, 1 0, 1

  28. Genetic Simulation COMMAND = ACTIVATE A A A P1 G1 G2 B P2 G3 G4 C P3 G5 P4 G6

  29. Genetic Simulation COMMAND = ACTIVATE B A B P1 G1 G2 B P2 G3 G4 C P3 G5 P4 G6

  30. Genetic Simulation COMMAND = ACTIVATE C A C P1 G1 G2 B P2 G3 G4 C P3 G5 P4 G6

  31. Pulse Encoding 1 1 1 0 0 0 Time Time Time Input Output

  32. Excitable protein output Protein Time Specification Design Modelling Implementation Testing/Validation

  33. Comparator Specifications 1: Strong Biobrick characterisation. 2: Precise relationship between coexpressed drug and reporter group. 3: Defined time in which to compute required pulse frequency.

  34. Timer Specifications Threshold Protein Time 1: Responsive to 1st light pulse only. 2: Restriction enzymes expressed at end of time period.

  35. Operation Summary Select desired drug INPUT MODULATION Select desired dosage COMPARATOR MODULATION Light Chemical

  36. Summary Light Receptor Start Timer Threshold detector Wavelength Processing Pulse Processing Restriction Enzyme Synthesis Drug Synthesis & Secretion

  37. Modelling considerations: Components • Protein controlled timer: • Simple logic gate representations • Timer block: • Rate of protein expression and degradation (ETH 07) • Threshold mechanism: “Schmitt trigger” (Taipei 07) • Encapsulation efficiency: • Particle size, morphology, swelling (Martins et al 2007) • Metabolic considerations: • Behaviour of bacteria inside the capsule (Wen-tao Qi et al. 2005) • Comparison with free in culture medium

  38. Model parameters • Protein controlled timer: - Light absorbance, pigment formation: Directly indicative of amount of protein present? • Encapsulation efficiency: - Diffusion of drug through capsule • Metabolic considerations: - Bacterial growth rate, population consumption

  39. Questions we would expect our models to answer • Protein controlled timer: - Obtain optimal input light conditions for protein degradation. • Encapsulation efficiency: - Find out optimal dimensions for maximal diffusion of substances through capsule. • Metabolic considerations: - Find optimal nutrient agar composition to obtain indication of bacterial survival.

  40. Summary • Solution: • User defined drug production for oral administration • 1 pill = 1 drug • High manufacturing cost • Variable peptide half life

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