A synthetic low-frequency mammalian oscillator

Area of Science:

• Synthetic Biology
• Chronobiology
• Molecular Systems Engineering

Background:

• Circadian clocks regulate essential physiological and behavioral processes across organisms.
• Dysregulation of circadian gene expression is linked to pathologies like tumors and metabolic disorders.
• Understanding complex gene network dynamics is crucial for biological insights and therapeutic development.

Purpose of the Study:

• To design and develop a novel, low-frequency synthetic mammalian oscillator.
• To achieve autonomous and robust transgene expression with circadian-like periodicity.
• To explore siRNA-mediated transcriptional silencing within a synthetic gene circuit.

Main Methods:

• Iterative cycles of mathematical model-guided design and experimental validation.
• Incorporation of intronically encoded small interfering RNA (siRNA) for gene silencing.
• Development of a tetracycline-dependent transactivator system for controlled gene expression.
• Real-time single-cell fluorescence microscopy of engineered Chinese Hamster Ovary (CHO-K1) cells.

Main Results:

• Successfully engineered a synthetic oscillator exhibiting autonomous and robust oscillatory behavior.
• Achieved a period of approximately 26 hours, mimicking natural circadian rhythms.
• Demonstrated real-time expression dynamics of a fluorescent reporter protein in single cells.
• Validated the efficacy of siRNA-based silencing within the synthetic gene circuit.

Conclusions:

• The novel synthetic oscillator provides a robust platform for studying biological rhythms.
• This design offers new insights into siRNA-mediated transcriptional silencing mechanisms.
• The developed oscillator holds potential for advancing synthetic gene circuit design and gene therapy applications.