The Universal PillThe Universal Pill

IGEM Presentation 17th July 09

James FieldDineka KhurmiNuri PurswaniKun Xue

Project DescriptionProject Description

Specification Design Modelling Implementation Testing/Validation

Problem for oral delivery of peptides

• 1 pill = 1 drug

• High manufacturing cost

• Variable peptide half life

Solution…

• User defined drug production…

The Universal PillThe Universal Pill

Specification Design Modelling Implementation Testing/Validation

• Multiple inputs enable drug selection

• Offers uniformity

• Direct packaging

• Fresh peptide production

• Dosage control

• Reduced loss of peptide

Capsule

Bacteria

Current MethodsCurrent Methods

Specification Design Modelling Implementation Testing/Validation

Peptide delivery Basic Mechanism Main Advantages Main DisadvantagesPolysaccharides Hydrogel

encapsulation- Safe- Biodegradable

- Hard to encapsulate peptides

Polyethylene glycol (PEG)

Conjugates to proteins forming amphiphilic complex

- Proteins not denatured- Can cross mucosa

- May alter biological function of protein

Liposomes Phospholipid bilayer carriers

- Targeting - Manufacturing

Symbiotic Microbe Secretion - Sustained drug production- Multiple drug outputs

- Difficult to regulate

Integrated SolutionIntegrated Solution

Specification Design Modelling Implementation Testing/Validation

Peptide delivery Basic Mechanism Main Advantages Main DisadvantagesPolysaccharides Hydrogel

encapsulation- Safe- Biodegradable

- Hard to encapsulate peptides

Polyethylene glycol (PEG)

Conjugates to proteins forming amphiphilic complex

- Proteins not denatured- Can cross mucosa

- May alter biological function of protein

Liposomes Phospholipid bilayer carriers

- Targeting - Manufacturing

Symbiotic Microbe Secretion - Sustained drug production- Multiple drug outputs

- Difficult to regulate

Chosen SolutionChosen Solution

Specification Design Modelling Implementation Testing/Validation

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

Encapsulate

Express

Kill

Release

Mechanism OverviewMechanism Overview• Polysaccharide encapsulation of chassis.• Peptide synthesis prior to consumption.

Light Trigger Logic CircuitLight Trigger Logic Circuit

Specification Design Modelling Implementation Testing/Validation

Drug 1

0

0

B

R

R = 0B = 0

R = 0B = 1

R = 1B = 0

R = 1B = 1

No production

Drug 1

Drug 2

Drug 3

Drug 2

Drug 3

Proposed ApplicationsProposed ApplicationsDisease General description Treatment drug Description

Diabetes Mellitus

Inability to metabolize glucose.

- GLP-1 Insulin production Glucagon production Beta cell mass and insulin gene expression Acid secretion and gastric emptying in the stomach Food intake by hunger

Haemophilia Hereditary disorder impairing body’s ability to control blood clotting or coagulation.

- Factor VIII in haemophilia A - Factor IX in haemophilia B

- Co-factor associated in the blood clotting cascade mechanism

Hay fever Allergic rhinitis frequently caused by pollen.

- Antigen - Reduces allergic reaction symptoms

Chassis CriteriaChassis Criteria

Testing/Validation

• Non pathogenic strain

• Large Biobrick availability

• Expertise in college

• Freeze dry

Chassis considerationsConsiderations

Chassis Non –pathogenic

Large bio-brickavailability

ExpertiseIncollege

Freeze dry

E.coli Yes Large Yes (most)

50% survival

B.subtilis Yes Few Yes est. 80% survival

S.cerevisiae Yes few Yes 10% survival

Capsule Design Capsule Design OverviewOverview

1) Encapsulation

2) Storage

3) Protein expression

4) Acid resistance

5) Release

1) Encapsulation1) Encapsulation

• enhances cell resistance to freezing and freeze-drying (for storage)

• added convenience and reduced packaging costs

• longer stability and viability during storage

Cell Encapsulation method

Mechanism Advantages Disadvantages

Alginate hydrogels

Sodium alginate forms solid gel with Ca2+

-mild-low cost

-leaking (for pure alginate)

Spray coating of fats

Dissolved in polymer and trapped in dried particle

- short contact time, good for labile particles

- Must be heat resistant for spray-dry encapsulation

Rennet- Gelling

Milk proteins form gels upon Rennet action

-milk is common food item

-storage problem

Encapsulation Method Encapsulation Method ComparisonComparison

Encapsulation detailsEncapsulation details

An in situ method for cultivating microorganisms using a double encapsulation techniqueEitan Ben-Dov1,2, Esti Kramarsky-Winter3,4 & Ariel Kushmaro1,5

2) Storage2) StorageShort term storage-up to a monthNutrient agarKeep in a sealable containerStorage in refrigerator

Long term storageInclusion of glycerol storage in freeze-dried formfreeze at -20°C or -80°C

Freeze drying

3) Protein expression

Transparency to light important for light inputs reaching cells

Alginate is transparent

Transparency type nutrient agar

Protein DepositionProtein Deposition

Proteins expressed are exported from the cell into the nutrient agar

Proteins stored in pores of nutrient agar until release

Exposure of bifidobacteria to simulated gastric juice at pH 2.0

Diameters of 40–80 μm - insignificant protection

1–3 mm - microspheres protected

entrapped cellsEncapsulation 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

4) Resistance to stomach 4) Resistance to stomach acidityacidity

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 1-3Disintegration times for alginate-

coating was 120 min

Resistance to AcidityResistance to Acidity

5) Release

Full degradation of alginate coat in intestines

Protein in nutrient agar now released

The Vision

LOAD PILLSELECT DRUGSELECT DOSECOMPETE

Black Box

Light

Chemical

BLACK BOX: Modules

Timer

Dose Control

Drug Control

Wavelength

Light Sensing

Peptide synthesis

Restriction enzyme

synthesis

Frequency

INPUT: Encoding with Light

Wavelength: Pulse:

Time

1

0

Drug Choice Dosage

Cph8

YcgF/YcgE

Wavelength Encoding

Output

Input

1, 0 1, 1 0, 1

A B C

Genetic Simulation

A

B

C

COMMAND = ACTIVATE A

P1 G1 G2

P2 G3 G4

P3 G5 P4 G6

A

Genetic Simulation

A

B

C

COMMAND = ACTIVATE B

P1 G1 G2

P2 G3 G4

P3 G5 P4 G6

B

Genetic Simulation

A

B

C

COMMAND = ACTIVATE C

P1 G1 G2

P2 G3 G4

P3 G5 P4 G6

C

Pulse Encoding

Output

Input

Time

1

0Time

1

0Time

1

0

Excitable protein output

Specification Design Modelling Implementation Testing/Validation

Time

Protein

Comparator Specifications1: Strong Biobrick characterisation.2: Precise relationship between

coexpressed drug and reporter group.3: Defined time in which to compute

required pulse frequency.

Timer Specifications1: Responsive to 1st light pulse only.2: Restriction enzymes expressed at

end of time period.

Time

Protein

Threshold

Operation Summary

Select desired drug

Light

Chemical

INPUT MODULATION

Select desired dosage COMPARATOR MODULATION

Summary

Light Receptor

Wavelength Processing

Pulse Processing

Start Timer

Drug Synthesis

& Secretion

Threshold detector

Restriction Enzyme

Synthesis

Modelling considerations: Modelling considerations: ComponentsComponents

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

Model parametersModel parametersProtein 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

Questions we would expect Questions we would expect our models to answerour 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.

SummarySummarySolution:

User defined drug production for oral administration

• 1 pill = 1 drug

• High manufacturing cost

• Variable peptide half life