To Rebehold the Cell: Natural and Synthetic Cell Cycles Through the Lenses of Macromolecular Motion

Summary

Humankind has a long-standing fascination with the idea of creating life. In this thesis, I started from the goal of creating a synthetic cell to hypothesise on what is needed to be defined as “alive”. A cell cycle was identified as a crucial component and the focus was put on the control of DNA replication of E. coli, which strikes an appealing compromise between simplicity and robustness. Of particular interest is the contribution that the poorly described titration of the replication initiator protein DnaA has in this control mechanism. A new experimental pipeline to perform single-particle tracking photoactivatable localisation microscopy was thus piloted and then applied to the study of DnaA behaviour in live E. coli cells. The results obtained in this way were employed to design and implement a synthetic genetic circuit enabling titration as the sole form of control of DNA replication in E. coli.