iGEM 2012 The bacterial Eyespot Bordeaux Team (France) Denis Dupuy - - PowerPoint PPT Presentation

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iGEM 2012 The bacterial Eyespot Bordeaux Team (France) Denis Dupuy - - PowerPoint PPT Presentation

May 21, 2023 44 likes •565 views

iGEM 2012 The bacterial Eyespot Bordeaux Team (France) Denis Dupuy Sophie Vaud Antoine Ribeiro Marie Beurthon-Aimar The 2012 Bordeaux Team Julie Di Martino Arnaud Frche Ccile Qur Jonathan Millet Mysterious fungus Christophe

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iGEM 2012

Bordeaux Team (France)

The bacterial Eyespot

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Denis Dupuy Marie Beurthon-Aimar

The 2012 Bordeaux Team

Arnaud Frèche Christophe Djemiel Sophie Vaud Antoine Ribeiro Julie Di Martino Cécile Quéré Jonathan Millet Mysterious fungus

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  • Introduction : The idea
  • Chapter 1 :The project
  • Chapter 2 : The simulation
  • Chapter 3 : The labwork
  • Conclusion : The prospect

Plan

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THE IDEA

Where our project came from ?

INTRODUCTION

When I went to school, they asked me what I wanted to be when I grew up. I wrote down ‘happy’. They told me I didn’t understand the assignment, and I told them they didn’t understand life. “ John Lennon

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SLIDE 5

Introduction

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

1

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Various pattern can be observed in nature

Zebra (Equus quagga) Leopard (Panthera pardus)

Introduction

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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Eyespots can be observed on some butterflies wings

Junonia coenia Inachis io

Introduction

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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A bacteria strain drawing concentric circles on a Petri dish

The idea

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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CHAPTER 1 THE PROJECT

How to make it real ?

“ A man who is no longer able to marvel at practically stopped living” Albert Einstein

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  • Bacterial Lawn (One enginereed strain)
  • 3 colored states (Operon-based differenciation)
  • Quorum-sensing signalisation

The project

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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Visible phenotype T

3 operons with :

  • A visible phenotype (LacZ/mCherry/GFP)

Operon-based cell Differenciation

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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Quorum- sensing activation Visible phenotype T

3 operons with :

  • A quorum-sensing activated Promoter

Operon-based cell Differenciation

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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Quorum- sensing activation Visible phenotype T

3 operons with :

  • A quorum-sensing signalling molecule producer

Signal- Producer

Operon-based cell Differenciation

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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  • 3 different types of quorum-sensing

Cell-to-cell communication

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion Petri dish Quantity of signalisation molecule

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  • 3 different types of quorum-sensing

Activation Threshold

Cell-to-cell communication

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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  • 3 different types of quorum-sensing

Cell-to-cell communication

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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  • 3 different types of quorum-sensing

Cell-to-cell communication

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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  • The necessity to use repressors to avoid

signalisation conflict

Double input

Cell-to-cell communication

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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Quorum- sensing activation Visible phenotype T

3 operons with :

  • A repressor of the 2 other operons
  • Two repressing sites in the promoter

Signal- Producer

Operon-based cell Differenciation

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

2 repressor binding site Repressor

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Operon 1

LasR-TetO LacIbox

RBS RBS RBS

LacZ LuxS CI T

Operon 4

LsrR LsrK LuxR T LasR

RBS RBS RBS RBS

Operon 2

pLsrA-CIbox LuxI mcherry LacI pTetR T

Operon 3

Luxbox- LacIbox

RBS RBS RBS

LasI TetR GFP CIbox T

RBS RBS RBS

Pc_Op4

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Operon 1

LasR-TetO LacIbox

RBS RBS RBS

LacZ LuxS CI T

Operon 2

pLsrA-CIbox LuxI mcherry LacI pTetR T

Operon 3

Luxbox- LacIbox

RBS RBS RBS

LasI TetR GFP CIbox T

RBS RBS RBS

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Regulation within the bacteria Operon I inhibits operons II and III

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Operon 1

LasR-TetO LacIbox

RBS RBS RBS

LacZ LuxS CI T

Operon 2

pLsrA-CIbox LuxI mcherry LacI pTetR T

Operon 3

Luxbox- LacIbox

RBS RBS RBS

LasI TetR GFP CIbox T

RBS RBS RBS

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Operon I activates operon II in the neighboring bacteria

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Active operon 1 CI Op 2 et 3 LuxS Active operon 2 LacI Op 1 et 3 LuxI

Operon-based cell Differenciation

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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Active operon 1 CI Op 2 et 3 LuxS Active operon 2 LacI Op 1 et 3 LuxI

Operon-based cell Differenciation

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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Active operon 1 CI Op 2 et 3 LuxS Active operon 2 LacI Op 1 et 3 LuxI

Operon-based cell Differenciation

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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Tet R Op 1 et 2 LasI Active operon 1 CI Op 2 et 3 LuxS Active operon 2 LacI Op 1 et 3 LuxI Active operon 3

Operon-based cell Differenciation

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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What can computer teach us ?

CHAPTER 2 THE SIMULATION

“They didn't know it was impossible, so they did it.“ Mark Twain

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Models our genetic regulatory network Includes eventual promoter leakage, mutation, etc… Python programming language

The simulation

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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The simulation

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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Everything is fine Operon I not signaling

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The simulation

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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Operon II not signaling Everything is fine

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The simulation

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion Operon III not signaling And promoter leakage Everything is fine

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Where are we now ?

CHAPTER 3 THE LABWORK

“ Science, my lad, is made up of mistakes, but they are mistakes which it is useful to make, because they lead little by little to the truth ” Jules Verne, Journey to the Center of the Earth

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  • Operon I : 3 assemblies left

Assembly

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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Assembly

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

  • Operon II complete

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Assembly

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

  • Operon III : 2 assemblies left

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Assembly

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

  • Operon IV : 3 assemblies left

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  • What is left ?

10/20 assemblies completed.

Assembly

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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  • Moving to a simpler system :

2 colored state ( 3 operons)

Assembly

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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What could came out of this project?

CONCLUSION THE PROSPECT

“ You should aim higher with your fantasies ” Lem, Veridian Dynamics

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  • Multiple Quorum-sensing Responsive

bacteria

LsrR LsrK LuxR T LasR RBS RBS RBS RBS Pc_Op4

Constitutive expression driven by biobrick J23100 aka « Pink Promoter »

The prospect

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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  • Enabling bacteria to have multiple functionnality

The prospect

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

Naive state 20

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  • Enabling bacteria to have multiple functionnality
  • E. Glowli Project

(2010 Cambridge)

The prospect

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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  • Enabling bacteria to have multiple functionnality

Eau d’E.coli Project (2006 MIT)

  • E. Glowli Project

(2010 Cambridge)

The prospect

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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  • Enabling bacteria to have multiple functionnality

Coliroid Project (2004 UCSF) Eau d’E.coli Project (2006 MIT)

  • E. Glowli Project

(2010 Cambridge)

The prospect

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

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  • Enabling bacteria to have multiple functionality
  • E. Glowli Project

(2010 Cambridge)

The prospect

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion « Reboot » function : Allow dedifferentiation Naive state

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1X signal

The prospect

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

If number of ring/circles dependant of the amount of initial signal :

  • Easy-to-read visible readout for chemical input

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1X signal 2X signal

The prospect

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

If number of ring/circles dependant of the amount of initial signal :

  • Easy-to-read visible readout for chemical input

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1X signal 2X signal 3X signal

The prospect

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

If number of ring/circles dependant of the amount of initial signal :

  • Easy-to-read visible readout for chemical input

21

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1X signal 2X signal 3X signal

The prospect

Introduction Chapter 1 Chapter 2 Chapter 3 Conclusion

Coupling with other iGEM project : Arsenic biosensor project (2006 Edinburgh)

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Image source :

http://www.thelensflare.com/imgs/eyespot-butterfly_47484.html http://artistjerrybennett.deviantart.com/art/Robot-and-Butterfly- 215933149 http://en.wikipedia.org/wiki/File:Zebra_in_Mikumi.JPG http://en.wikipedia.org/wiki/File:Slleo1.jpg http://fr.wikipedia.org/wiki/Fichier:Inachis_io_LC0131.jpg http://fr.wikipedia.org/wiki/Fichier:Junonia_coeniaPCCA20051015- 1147B.jpg

Annexe

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The sponsors

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Thank you for your time !

The world is my country, science is my religion.‘ – Christiaan Huygens, Dutch Physicist (1629-1695)