Students : Aimeric Agaoua, Lambert Antoni, Clara Bouyx, Vincent - - PowerPoint PPT Presentation

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Students : Aimeric Agaoua, Lambert Antoni, Clara Bouyx, Vincent - - PowerPoint PPT Presentation

Jan 28, 2024 34 likes •314 views

Students : Aimeric Agaoua, Lambert Antoni, Clara Bouyx, Vincent Castel, Alexia Satouf, and Romain Veillard Introduction During growth, every single cell follows its own cell cycle Introduction During growth, every single cell follows its own

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Students : Aimeric Agaoua, Lambert Antoni, Clara Bouyx, Vincent Castel, Alexia Satouf, and Romain Veillard

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Introduction

During growth, every single cell follows its own cell cycle

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During growth, every single cell follows its own cell cycle

Introduction

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During growth, every single cell follows its own cell cycle

Introduction

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Modelling of our integrated system

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Modelling of our integrated system

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We need to control the system : stability of each components speed of system

Modelling of our integrated system

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We need to control the system : stability of each components speed of system We need traffic lights

Modelling of our integrated system

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Initial unsynchronised bacteria

Our goal : synchronize a culture of E.coli cells

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Initial unsynchronised bacteria

Our goal : synchronize a culture of E.coli cells

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In labs, physical and chemical treatments exist to synchronise the cell cycle

Long and expensive manipulations

Hydroxyurea

Existing solutions

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To develop a new biological solution where bacteria synchronize automatically at a whole culture scale To model an oscillating system using a negative feedback loop

Our Goal

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Three components :

  • A signaling molecule : serine
  • A coordinator : CheA-SH3
  • A division inhibitor : ppGpp

RelA Mesh1

System for bacteria synchronisation

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Serine

3-PG ∆ sda B ∆ sda C

PcusC

Serine

serA (short)

SdaC Pyruvate SdaB Serine Serine

P CusR

stab SerA

Serine, a signalling molecule

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Continuous increase of extracellular [Serine] Serine

3-PG ∆ sda B ∆ sda C

PcusC

Serine

serA (short)

SdaC Pyruvate SdaB Serine Serine

P CusR

stab SerA

Serine, a signalling molecule

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P CheA

Chemotaxis

P

CheY

CheA‐SH3, a coordinator

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P CheA

Chemotaxis

P

CheY

CheA‐SH3, a coordinator

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P

CusR

P CheA

SH34 LZA sh3pep-l-lza

P CheA

Chemotaxis

P

CheY

CheA‐SH3, a coordinator

Ressource : Whitaker et al. 2012

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CusR

Pconstitutive

cheA RBS T RBS RBS cusR-l-lza

sh3pep-l-lza

CheA

P

CusR

P

CusR

P CheA

SH34 LZA

[Serine]

CheA‐SH3, a coordinator

Tsr receptor

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CusR

Pconstitutive

cheA RBS T RBS RBS cusR-l-lza

sh3pep-l-lza

CheA CusR CusR

P

CheA

P

CusR

P

CusR

P CheA

SH34 LZA

[Serine] [Serine]

CheA‐SH3, a coordinator

Tsr receptor

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Tsr receptor CusR

Pconstitutive

cheA RBS T RBS RBS cusR-l-lza

sh3pep-l-lza

CheA CusR CusR

P

CheA

P

CusR

P

CusR

P CheA

SH34 LZA

[Serine] [Serine] Modulation of CusR phosphorylation as a function of serine level

CheA‐SH3, a coordinator

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Growth of cells in the presence of Ag 2 or 3 CusR boxes sequester the endogenous CusR Normal detoxification of the cells CusR binds to CusR box sequences in reponse to metallic stress (Cu, Ag).

Time (min) OD (600 nm)

Validation of our part BBA_K1349002

(CusR box sequence)

No box CusR box x1 CusR box x2 CusR box x3

pUC + (CusR box)n

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CusR phosphorylated RFP expression Overexpression of RelA P CusR

mesh1 relA T T rfp gfp T T

RFP

ppGpp Hold on G0 phase

RelA

Increase of intracellular ppGpp level Cell cycle stopped for all bacteria

PcusC Pconstitutive

Increase of intracellular ppGpp concentration to STOP cell division

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CusR unphosphorylated GFP expression Overexpression of Mesh1

Decrease of intracellular ppGpp level Division process restarts

CusR

mesh1 relA T T rfp gfp T T

PcusC Pconstitutive

ppGpp

Go to S phase

Mesh1

GFP

Decrease of intracellular ppGpp concentration to RESTART cell division

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Mutant unable to synthetize RelA, and growing very slowly. Complementation with our part Bacteria normal growth is restored. Cell division control thanks to [ppGpp] modulated by RelA and Mesh1

Validation of our part BBa_K1349001 (RelA coding sequence)

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A computer simulation of the designed system shows that CusR, and ppGpp can

  • scillate as the system grows and serine accumulates

Our system seems to be the expected one : It oscillates with cell division

Modelling, the return : Estimation of parameters

SerA Serine in Serine

  • ut

CheA CusR-P ppGpp

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Our project, cells division control thanks to : Serine as a signalling molecule CheA-SH3 to coordinate the system Modulation of intracellular ppGpp concentration Our achievements, two caracterized BioBrick Part : Submission of 8 BioBrick Parts Experimental validation of one new BioBrick Part :

BBA_K1349002 (CusR box sequence)

Improvement of the function and characterization of an existing BioBrick Part :

BBa_K1349001 (RelA coding sequence) in amelioration of Bba_K639001

Our prospects, a conference at Aix-Marseille University : Presentation of our project and our experience feedback

Conclusion and prospects

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Thank you for your attention !

Supervisors : Gaël Chambonnier, Laëtitia Houot, Sandra Michel-Souzy, and James Sturgis Advisors : Aurélie Dobric Collaborators : Dr. E. Bouveret (LISM, Marseille) Ressources : Dr. J. Dueber (Berkeley University, USA), and Dr. B. Field (CNRS, Marseille)