Abstract

Circadian rhythms are present in diverse organism, from bacteria to mammals. Although they appear independently throughout evolution, the molecular bases governing them are conserved. The central circadian oscillator is composed of a transcriptional-translational feedback loop (TTFL), where the negative element inhibits directly the positive element, which promotes its transcription. In Neurospora crassa the negative element is the protein FREQUENCY, encoded by the gene frq, and the positive element the White Collar complex (WCC). In addition, the rhythmic information is transmitted through a transcriptional cascade causing the rhythmic expression of genes controlled by the clock (ccgs). To improve our knowledge in the genetic topological plasticity of the clock, we used transcriptional rewiring technics, a synthetic biology approach, to generate new circuitry topologies of the central clock. We generate Hybrid Oscillators (HOs) changing the promoter of frq with the promoter of a ccg and evaluated the capacity of the system to generate and sustain rhythms even the TTFL architecture is modified. Using this approach, we demonstrated the ability of the core oscillator to sustain rhythms, even when the evolutionary conserved core oscillator is dramatically modified. The HO with better performance displays circadian oscillations which are subjected to FRQ dynamics and is temperature compensated. Interestingly, the light response and phase determination are different in the HO. Finally, we confirmed that the HO has an extended TTFL architecture where at least five additional transcriptional regulators are now part of the core circadian oscillator