Transcript Production of the Antimalarial Drug Precursor

The Universal Pill
IGEM Presentation
17th 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…
Specification
Design
Modelling
Implementation
Testing/Validation
The Universal Pill
• Multiple inputs enable drug selection
• Offers uniformity
• Direct packaging
• Fresh peptide production
Capsule
• Dosage control
Bacteria
• Reduced loss of peptide
Specification
Design
Modelling
Implementation
Testing/Validation
Current Methods
Peptide delivery
Polysaccharides
Basic Mechanism
Hydrogel
encapsulation
Polyethylene glycol
(PEG)
Conjugates to
- Proteins not
proteins forming
denatured
amphiphilic complex - 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
Specification
Design
Modelling
Main Advantages
- Safe
- Biodegradable
Implementation
Main Disadvantages
- Hard to
encapsulate
peptides
Testing/Validation
Integrated Solution
Peptide delivery
Polysaccharides
Basic Mechanism
Hydrogel
encapsulation
Polyethylene glycol
(PEG)
Conjugates to
- Proteins not
proteins forming
denatured
amphiphilic complex - 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
Specification
Design
Modelling
Main Advantages
- Safe
- Biodegradable
Implementation
Main Disadvantages
- Hard to
encapsulate
peptides
Testing/Validation
Chosen Solution
Capsule
Polysaccharide encapsulation
of chassis
Bacteria
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
Mechanism Overview
Encapsulate
Express
• Polysaccharide
encapsulation of chassis.
• Peptide synthesis prior
to consumption.
Kill
Release
Specification
Design
Modelling
Implementation
Testing/Validation
Light Trigger Logic Circuit
0
B
R
0
Drug 1
Drug 2
Drug 3
Specification
Design
Modelling
R=0
B=0
No
production
R=0
B=1
Drug 1
R=1
B=0
Drug 2
R=1
B=1
Drug 3
Implementation
Testing/Validation
Proposed Applications
Disease
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 Criteria
• Non pathogenic strain
• Large Biobrick availability
• Expertise in college
• Freeze dry
Testing/Validation
Chassis considerations
Considerations
Chassis
Non –
pathogenic
Large
bio-brick
availability
Expertise Freeze dry
In
college
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 Overview
1) Encapsulation
2) Storage
3) Protein expression
4) Acid resistance
5) Release
1) Encapsulation
• enhances cell resistance to freezing and freeze-drying
(for storage)
• added convenience and reduced packaging costs
• longer stability and viability during storage
Encapsulation Method Comparison
Cell
Encapsulation
method
Mechanism
Advantages
Disadvantages
Alginate
hydrogels
Sodium
alginate forms
solid gel with
Ca2+
-mild
-low cost
-leaking (for
pure alginate)
Spray coating Dissolved in
of fats
polymer and
trapped in dried
particle
- short
contact
time, good
for labile
particles
- Must be
heat resistant
for spray-dry
encapsulation
RennetGelling
-milk is
common
food item
-storage
problem
Milk proteins
form gels upon
Rennet action
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
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
Freeze drying
3) Protein expression

Transparency to light important for light
inputs reaching cells

Alginate is transparent

Transparency type nutrient agar
Protein Deposition

Proteins expressed are exported from
the cell into the nutrient agar

Proteins stored in pores of nutrient agar
until release
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
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

5) Release

Full degradation of alginate coat in
intestines

Protein in nutrient agar now released
The Vision
LOADDRUG
PILL
SELECT
SELECT
COMPETE
DOSE
Black Box
Light
Chemical
BLACK BOX: Modules
Drug Control
Dose Control
Light Sensing
Wavelength
Peptide
synthesis
Frequency
Timer
Restriction
enzyme
synthesis
INPUT: Encoding with Light
Wavelength:
Pulse:
Cph8
1
YcgF/YcgE
Drug Choice
0
Time
Dosage
Wavelength Encoding
Input
A
B
C
1, 0
1, 1
0, 1
Output
Genetic Simulation
COMMAND = ACTIVATE A
A
P1
G1
G2
P2
G3
G4
P3
G5
A
B
C
P4
G6
Genetic Simulation
COMMAND = ACTIVATE B
A
P1
G1
G2
P2
G3
G4
P3
G5
B
B
C
P4
G6
Genetic Simulation
COMMAND = ACTIVATE C
A
P1
G1
G2
P2
G3
G4
P3
G5
C
B
C
P4
G6
Pulse Encoding
Input
Output
1
1
1
0
0
0
Time
Time
Time
Excitable protein output
Protein
Time
Specification
Design
Modelling
Implementation
Testing/Validation
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.
Timer Specifications
1: Responsive to 1st light pulse only.
2: Restriction enzymes expressed at end of time
period.
Threshold
Protein
Time
Operation Summary
Select desired drug
INPUT MODULATION
Select desired dosage
COMPARATOR
MODULATION
Light
Chemical
Summary
Light Receptor
Start Timer
Wavelength
Processing
Threshold detector
Pulse Processing
Restriction Enzyme
Synthesis
Drug Synthesis
& Secretion
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
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
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.
Summary
Solution:
User defined drug production for oral administration
• 1 pill = 1 drug
• High manufacturing cost
• Variable peptide half life