Insights into a clock's fidelity through vesicular encapsulation

Area of Science:

• * Systems Biology
• * Synthetic Biology
• * Chronobiology

Background:

• * Cyanobacteria, like *Synechococcus elongatus*, exhibit highly precise circadian rhythms in tiny volumes.
• * The KaiABC post-translational oscillator (PTO) drives these rhythms.
• * Understanding the mechanisms of circadian fidelity in confined cellular environments is crucial.

Purpose of the Study:

• * To investigate the mechanistic basis of circadian rhythm fidelity in reconstituted KaiABC PTOs.
• * To explore the influence of protein concentration and vesicle size on PTO performance *in vitro*.
• * To model the factors contributing to circadian clock accuracy in cyanobacteria.

Main Methods:

• * Reconstitution of the KaiABC PTO in cell-mimetic giant unilamellar vesicles (GUVs).
• * Encapsulation of PTO proteins with physiologically relevant variation.
• * Single-vesicle tracking of circadian rhythms using fluorescently labeled KaiB and confocal microscopy.
• * Mathematical modeling of PTO behavior and cyanobacterial clock components.

Main Results:

• * Circadian rhythms were successfully generated and measured in thousands of GUVs over several days.
• * PTO fidelity decreased with lower protein concentrations and in smaller GUVs.
• * KaiB localized to GUV membranes, mimicking its behavior in cyanobacteria.
• * Mathematical modeling suggested high expression of PTO and buffering proteins (CikA, SasA) enhance fidelity.

Conclusions:

• * This study provides the first experimental demonstration of synthetic cells autonomously keeping circadian time.
• * High protein expression levels and larger cellular volumes are key for robust circadian rhythm fidelity.
• * The transcription-translation feedback loop (TTFL) plays a minor role in fidelity but is essential for synchrony under constant conditions.
• * The *in vitro* reconstitution approach offers insights into collective biological behaviors like phase separation.