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Related Experiment Video

Updated: Jun 9, 2025

Rapid Encapsulation of Reconstituted Cytoskeleton Inside Giant Unilamellar Vesicles
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Insights into a clock's fidelity through vesicular encapsulation.

Alexander Zhan Tu Li, Andy LiWang, Anand Bala Subramaniam

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    This study reconstitutes the KaiABC circadian clock in artificial cells, revealing that high protein levels and larger cell size enhance rhythm fidelity. This work demonstrates synthetic cells can autonomously keep time.

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    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.