The Universal Pill

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

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
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Light Trigger Logic Circuit
0
Drug 1
R 0 B 0
No production
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
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Proposed Applications
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Chassis Criteria

• Non pathogenic strain
• Large Biobrick availability
• Expertise in college
• Freeze dry

Testing/Validation
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Chassis considerations
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Capsule Design Overview

• Encapsulation
• Storage
• Protein expression
• 4) Acid resistance
• 5) Release

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1) Encapsulation

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

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Encapsulation Method Comparison
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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
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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 -20C or -80C

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Freeze drying
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3) Protein expression

• Transparency to light important for light inputs
  reaching cells
• Alginate is transparent
• Transparency type nutrient agar

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Protein Deposition

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

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4) Resistance to stomach acidity

• Exposure of bifidobacteria to simulated gastric
  juice at pH 2.0
• Diameters of 4080 µm
• - insignificant protection
• 13 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
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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

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5) Release

• Full degradation of alginate coat in intestines
• Protein in nutrient agar now released

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The Vision
LOAD PILL
SELECT DRUG
SELECT DOSE
COMPETE
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Black Box
Light
Chemical
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BLACK BOX Modules
Drug Control
Dose Control
Light Sensing
Frequency
Timer
Wavelength
Peptide synthesis
Restriction enzyme synthesis
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INPUT Encoding with Light
Wavelength
Pulse
Cph8
1
0
YcgF/YcgE
Time
Drug Choice
Dosage
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Wavelength Encoding
Input
A
B
C
Output
1, 0
1, 1
0, 1
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Genetic Simulation
COMMAND ACTIVATE A
A
A
P1 G1 G2
B
P2 G3 G4
C
P3 G5 P4 G6
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Genetic Simulation
COMMAND ACTIVATE B
A
B
P1 G1 G2
B
P2 G3 G4
C
P3 G5 P4 G6
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Genetic Simulation
COMMAND ACTIVATE C
A
C
P1 G1 G2
B
P2 G3 G4
C
P3 G5 P4 G6
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Pulse Encoding
Input
Output
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Excitable protein output
Protein
Time
Specification
Design
Modelling
Implementation
Testing/Validation
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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.

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Timer Specifications

• 1 Responsive to 1st light pulse only.
• 2 Restriction enzymes expressed at end of time
  period.

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Operation Summary
Select desired drug
INPUT MODULATION
Select desired dosage
COMPARATOR MODULATION
Light
Chemical
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Summary
Light Receptor
Start Timer
Threshold detector
Wavelength Processing
Pulse Processing
Restriction Enzyme Synthesis
Drug Synthesis Secretion
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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

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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

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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.

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Summary

• Solution
• User defined drug production for oral
  administration
• 1 pill 1 drug
• High manufacturing cost
• Variable peptide half life