Dottorato in GENETICA E BIOLOGIA MOLECOLARE

Titolo della tesi: Optogenetics in bacteria: engineering optical control in synthetic genetic oscillators

Light is emerging as a powerful tool to investigate and control natural and synthetic cellular processes. In this thesis, we modeled, developed, and characterized a synthetic genetic oscillator controllable through light inputs. We started from the repressilator, a synthetic oscillatory circuit that was a milestone in the field of synthetic biology, and a light-driven gene expression system, CcaS-CcaR. We designed a mathematical model to represent our ideal outcome circuit and then refined the model according to the emerging experimental data. To build the circuit, we faced the problem of correctly interfacing two genetic systems and solved it through genetic engineering. While doing this, we paid attention to the use of modular and non-case-specific engineering steps in order to develop a possible guideline for adding an optogenetic module to a pre-existing circuit. The result is the optorepressilator. To analyze the circuit’s behavior, we combined population and single-cell experiments with computational simulations. We demonstrated that a population of optorepressilators can be synchronized and entrained using light. We analyzed the single-cell output to light inputs, examining the phase response of the system. We characterized the response to detuning inputs, highlighting how entrainment influences the system’s frequency and creates dierent synchronization states. This analysis highlighted our model’s performance in interpreting and predicting experimental data, recapitulating several physics phenomenologies. These results deepened our knowledge of the behavior of genetic oscillators, with similarities to the human circadian clock. We showed optogenetics’ versatility in controlling a genetic network and how theory and experiments can interplay in this effort. Furthermore, we enriched synthetic biology’s toolbox with a light-driven oscillatory circuit and its theoretical framework. Finally, we show how light can be applied to other cellular processes, such as the investigation of flagellar bundling dynamics.