Generation of hybrid circadian oscillators through transcriptional rewiring

Circadian rhythms are self-autonomous rhythms generated at the cellular level with a period close to 24 hours, present in diverse organism from bacteria to mammals. Although they have appeared independently throughout evolution, their design principles and circuitry logic are conserved. Thus, the central circadian oscillator is composed of a transcriptional-translational negative feedback loop (TTFL), where the negative element directly inhibits the positive element that promotes its transcription.

To extend our knowledge of the topological properties of the highly-conserved clock TTFL, in this thesis we used transcriptional rewiring technics, a synthetic biology approach, to generate new circuitry topologies of the Neurospora central oscillator. We generated Hybrid Oscillators (HOs) changing the promoter of the main clock component by the promoter of a downstream clock-controlled gene and evaluated the capacity of the system to generate and sustain rhythms.

Using this approach, we demonstrated the ability of the core oscillator to sustain rhythms, despite the fact that the central topology and connectivity have been dramatically modified. The HO with better performance, HO-10, displays circadian oscillations where the period is controlled by posttranslational events, and is temperature compensated. Interestingly, in HO-10 the response to light and phase determination are different. We confirmed that HO-10 has an extended TTFL architecture where at least five additional transcriptional regulators are now part of the core circadian oscillator.

In toto, these results confirm that alternative circuit topologies are compatible with clock function, yet such designs compromise robustness to genetic perturbations.

• Nombre: Alejandra Goity Falconi
• Laboratorio: Cronobiología
• Mención: Genética Molecular y Microbiología
• Director Tesis: Luis Larrondo