Dr. Coli The bacterial drug delivery system

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THE DR. COLI PROJECT BELGIUM - LEUVEN

Leuven - BELGIUM

Multidisciplinary team

Dr. Coli Dr. Coli

The bacterial drug The bacterial drug

delivery system delivery system

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Different domains of Synthetic Biology

Food or energy

Environment

Manufacturing

Bioreactors

Smart materials

Sensors

….

Project Situation

Health or medicine

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Project Situation

Problems with conventional

therapy

Solution:

Selfregulating drugs: Dr. Coli

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Project Specifications

Characteristics

Sensing – Producing – Eliminating

Filter: non-consistent signal kicked out

Modeling

Integrative model

Wet lab

Reuse of existing parts and devices

New devices and characterization

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Our Results

Characteristics

Complete design of devices

Modeling

Project fully simulated

Sensitivity analysis successful robust model

New toolbox for Matlab: Multicell toolbox

Wet lab

6 new parts and 4 new devices built

1 part and 2 devices fully characterized

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Overview

7 subsystems

Global system

Modeling

Memory Input

Filter

Reset

Cell Death

Output

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Input - Output

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Dummy input

Dummy output

GFP-LVA

Extensive

characterization

Half life reduction:

35x

Input - Output

Comparison of GFP to GFP-LVA

1 10 100 1000

relative fluorescence (%)

GFP GFP-LVA

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InverTimer & Cell Death

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No disease marker → Dr. Coli dies

Inverter

Timer molecules (HSL)

Suicide gene

InverTimer & Cell Death

Input InverTimer Cell death

0 1 1

1 0 0

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Reset

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Reset

Renewed disease marker signal

→ timer reset

Lactonase

Timer molecule (HSL) degradation

Timer reset

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Memory

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Memory

Production problem

One time « ON » switch

AND Reset

InverTimer

Memory Cell Death

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Filter

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Filter

Coherent feedforward loop with AND-gate

X: short-lived

Y: longer-lived

AND

Input X Y

Z

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Modeling, a necessity

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Modeling, a necessity

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Dr. Coli @ work

Five different phases:

1. No disease signal 2. First disease signal

3. Temporarily/locally cured 4. Patient relapse

5. Patient cured

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@ work 1: No Disease Signal

Input Output

Filter Reset Memory

OFF

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Simulation 1: No Disease Signal

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@ work 2: 1 ^st Disease Signal

ON

Reset Filter

Memory

Input Output

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Simulation 2: 1 ^st Disease Signal

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@ work 3: Temp/locally Cured

Output

ON

Filter Memory

Input

Reset

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Simulation 3: Temp/locally Cured

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@ work 4: Patient Relapse

Output

ON

Filter Memory

Input

Reset

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Simulation 4: Patient Relapse

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@ work 5: Patient Cured

Output

ON

Filter Memory

Input

Reset

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Simulation 5: Patient Cured

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Optimizing our model

Adaptations

Robustness

Multicell system

Toolbox interacts with SimBiology

Memory & timer inheritance

HSL signaling molecules

Modeling

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Modeling Modeling

Documented parameters

Fully working integrative robust model

Multicell toolbox

Open Source Wiki Open Source Wiki

Daily updates

NEW PARTS NEW DEVICES

GFP-LVA Input

T7 Output

T7-UmuD Cell Death

asLuxI InverTimer

CcdB Reset

Hybrid Prom. Memory Filter

S S

P P

P P P P S S

S S S S

Ethics &

Human Practice Human Practice Wet Lab

Wet Lab

Achievements

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P: Partially finished

P: Partially finished S: Sequenced S: Sequenced

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Many thanks to:

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Leuven

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Input - Output

Input: disease marker

Dummy Input: TetR ~ aTc

Output: drugs

Dummy Output: GFP-LVA

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GFP-LVA

GFP-LVA vs GFP

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InverTimer & Cell Death

InverTimer

Cell Death

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Diffusion of Timermolecule

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Reset

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Filter

AND Input RiboKey T7 RNAPol

Z

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Memory

One time « ON » switch

Two repressors: R ₁ and R ₂

« OFF » R ₁ low, R ₂ high

« ON » R ₁ high, R ₂ low

« ON » stable

« OFF » timer does not count

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Multi Cell System

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