The Programming of a Cell By L Varin and N Kharma Biology and Computer Engineering Departments...

The Programming of a Cell

By L Varin and N KharmaBiology and Computer Engineering DepartmentsConcordia University

Artificial Life GroupThe Programming of a Cell

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Motivation • Cells have advantages

over silicon:• They have/can have built-in

interfaces, to sense and produce many biological substances

• They are easy to mass produce, store and distribute

• They are generally more robust than man-made systems

• They are optimizable via (real) evolution


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Motivation – precisely

• The aim is to produce a cell that implements a configurable Boolean logic function in 2 var’s• Ultimately, we would like to use intercellular signalling to compile larger circuits using many smaller ones

A mechanical AND gate


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Outline

• Motivation & Outline• Biological Background• Problem Statement• Alternative Methods• Biological Realization• Practical Significance


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Biological Background:Flow of genetic information

DNA RNA Protein

We can easily manipulate DNA

Transcription Translation

Gene expression


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Biological Background:Gene expression (promoters)

+1

-10Box

TATAA

-35Box

TTGTCARNA

Core promoter = Binding site for RNA Polymerase

In this configuration transcription is ON

RNA Pol


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+1

-10box

-35box

Biological Background:Gene expression (promoters)

operator

R

R = Repressor

In this configuration RNA Polymerase cannot bindtranscription is OFF

X


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• Construct a promoter• Insert an operator• Select a coding sequence (output)

Biological Background:Gene expression (synthetic gene)

-10box

-35box

operator

Modular structure

Output


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Biological Background:Biological regulatory network

• The lactose operon of E. coli

R

lacIrepressor R

-35 O -10

Transcription is OFF

Active repressor

X


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Biological Background:Biological regulatory network

• The lactose operon of E. coli

R

lacIrepressor

-35 O -10

Transcription is ON

Inactiverepressor

= inducer (lactose)

RNA Pol

X


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Biological Background:Artificial regulatory network

• Select an output gene• Select a promoter• Select an operator-repressor

system• Assemble the parts together


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Green Fluorescent protein

C1 repressor

promoter

Lac promoterRepressed by lac repressor

Repressed by C1 repressor

lacI

ON

OFF

- lactose

X


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Green Fluorescent protein

C1 repressor

promoter

Lac promoterRepressed by lac repressor

Repressed by C1 repressor

OFF

R+ lactose

X

X

C1

C1



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Problem Statement ++

• Synthesize a cell that can be configured to implement any one of 16 different Boolean functions in 2 variables

• Such a project will involve 4 phases:1 Designing a regulatory

network2 Constructing a

configurable cell3 Configuring the cell4 Using the cell


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Methodology 1: Using Repressilators

A BOutput

R1

R2

Anti-sense DNA


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Methodology 1: Full PictureA B’

Output

R3

R4

Anti-sense DNA

A BOutput

R1

R2

Anti-sense DNA

A’ B’Output

R5

R6

Anti-sense DNA

A’ BOutput

R7

R8

Anti-sense DNA


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Methodology 2: Using Excision

• A Boolean function in 2 variables has 16 possible truth tables

• They all involve 1-4 (3) different terms of 2 variables

A B Output

00 ?

01 ?

11 ?

10 ?


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Methodology 2: Chosen Path• Design

A regulatory network implementing the 4 terms and allowing for subsequent excision of any term

• ConstructThe regulatory network by embedding 4 gene networks

corresponding to the 4 terms in a real organism (e.g. e.coli)

• ConfigureThe cell by excising those gene networks corresponding to

the unwanted terms

• UseThe configured cell by adding the inducers (variables) it is

designed to respond to, and monitoring the output


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• 2 variables A and B• A = lactose• B = arabinose

• 1 promoter • 4 repressors

• 1 ouput gene (Green Fluorescent Protein)

• 4 terms (A B), (A B), (A B), (A B)

Biological Realization


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(A B)Output (GFP)

= Lac operon operator (bound by LacI repressor)

= Arabinose operon operator (bound by AraR repressor)

In the absence of A and B

Output (GFP)LacI AraR X


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(A B)Output (GFP)

In the presence of A and B ( lactose and arabinose)

Output (GFP)

LacI AraR

LacI AraR

X

XX


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(A B)Output (GFP)

= Pr operator (bound by C1 repressor)

In the absence of A and in presence of B

Output (GFP)

XC1

LacI

= Arabinose operon operator (bound by AraR repressor)

AraR

X


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(A B)Output (GFP)

= lactose operon operator (bound by lacI repressor)

In the presence of A and in absence of B

Output (GFP)

XCRO

= Prm operator (bound by CRO repressor)

LacI

X

AraR


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(A B)Output (GFP)

= Pr operator (bound by C1 repressor)

In the absence of A and in absence of B

Output (GFP)

XCRO

= Prm operator (bound by CRO repressor)

AraR

C1

LacI X


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(A B)

(A B)

(A B)

(A B)

Output (GFP)

Output (GFP)

Output (GFP)

Output (GFP)


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Practical Significance

Limited ConfigurableDecision Logic

Inputs tied to a particular application: lactose, arabinose etc.

Outputs tied to a particular application: GFP etc.


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Practical Significance

Extended Decision Logic

Input Interface

Output Interface

Application-specific inputs

Application-specific outputs

Standardized Signals


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Summary

• A specific Boolean logic function in 5 variables has been recently realized in living cells, but never a configurable bio-logic device theoretic value

• We believe we have found a simple means of realizing a configurable 2-input Boolean function in an e.coli cell simple methodology

• Both the logic functionality and the practical value of the work can be considerably enhanced with the use of intercellular signaling broader vision

• First experiments (for the Method 2) will start in January 2008 and we’ll update you!

The Programming of a Cell By L Varin and N Kharma Biology and Computer Engineering Departments...

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