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

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 cell cycle

Introduction During growth, every single cell follows its own cell cycle

Modelling of our integrated system

Modelling of our integrated system

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

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

Our goal : synchronize a culture of E.coli cells Initial unsynchronised bacteria

Our goal : synchronize a culture of E.coli cells Initial unsynchronised bacteria

Existing solutions In labs, physical and chemical treatments exist to synchronise the cell cycle Hydroxyurea Long and expensive manipulations

Our Goal 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

System for bacteria synchronisation Three components : ● A signaling molecule : serine ● A coordinator : CheA-SH3 Mesh1 RelA ● A division inhibitor : ppGpp

Serine, a signalling molecule Serine Serine Serine SdaC 3-PG Serine SdaB P CusR SerA Pyruvate P cusC ∆ sda B serA (short) stab ∆ sda C

Serine, a signalling molecule Serine Continuous increase of Serine extracellular [Serine] Serine SdaC 3-PG Serine SdaB P CusR SerA Pyruvate P cusC ∆ sda B serA (short) stab ∆ sda C

CheA ‐ SH3, a coordinator P CheA P CheY Chemotaxis

CheA ‐ SH3, a coordinator P CheA P CheY Chemotaxis

CheA ‐ SH3, a coordinator P CheA SH3 4 P CheA sh3pep-l-lza P CheY P LZA CusR Chemotaxis Ressource : Whitaker et al. 2012

CheA ‐ SH3, a coordinator [Serine] Tsr receptor P CusR P CheA P CusR SH3 4 LZA CheA CusR P constitutive T RBS cheA RBS RBS cusR-l-lza sh3pep-l-lza

CheA ‐ SH3, a coordinator [Serine] [Serine] Tsr receptor P CusR CusR P CheA CheA P P CusR CusR SH3 4 LZA CheA CusR P constitutive T RBS cheA RBS RBS cusR-l-lza sh3pep-l-lza

CheA ‐ SH3, a coordinator [Serine] [Serine] Tsr receptor P CusR CusR P CheA CheA P P CusR CusR Modulation of CusR phosphorylation as a function of serine level SH3 4 LZA CheA CusR P constitutive T RBS cheA RBS RBS cusR-l-lza sh3pep-l-lza

Validation of our part BBA_K1349002 (CusR box sequence) CusR binds to CusR box sequences in reponse to metallic stress (Cu, Ag). pUC + (CusR box) n OD (600 nm) No box CusR box x1 CusR box x2 CusR box x3 Time (min) Growth of cells in the presence of Ag 2 or 3 CusR boxes sequester the endogenous CusR Normal detoxification of the cells

Increase of intracellular ppGpp concentration to STOP cell division Hold on G0 phase ppGpp RelA P constitutive P cusC relA mesh1 T T gfp rfp T T P CusR RFP CusR phosphorylated Increase of intracellular ppGpp level RFP expression Cell cycle stopped for all bacteria Overexpression of RelA

Decrease of intracellular ppGpp concentration to RESTART cell division Go to S phase ppGpp P constitutive Mesh1 P cusC relA mesh1 T T gfp rfp T T GFP CusR CusR unphosphorylated Decrease of intracellular ppGpp level GFP expression Division process restarts Overexpression of Mesh1

Validation of our part BBa_K1349001 (RelA coding sequence) Cell division control thanks to [ppGpp] modulated by RelA and Mesh1 Mutant unable to synthetize RelA, and growing very slowly . Complementation with our part Bacteria normal growth is restored .

Modelling, the return : Estimation of parameters CusR -P SerA Serine ppGpp in Serine CheA out A computer simulation of the designed system shows that CusR, and ppGpp can oscillate as the system grows and serine accumulates Our system seems to be the expected one : It oscillates with cell division

Conclusion and prospects 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

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)