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Spindle-deficient protein 5 derived from C.elegans is able to assemble into condensates that built a separate phase from the surronding cytosol, therefore emulating a reaction compartment. Fusion proteins will also be confined to this organelle.

SPD 5 proteins assemble into phase-separated condensates in the cytosol, building a dynamic artificial organelle. Fusion proteins of SPD5 will be confined inside the organelle and separated from the all other components inside the cell. Due to the lack of a membrane, all other molecules can freely move in and out of the organelle.

In C. elegans, SPD5 , forms condensates that aid the organization of microtubule arrays in the centrosome.

By expressing a SPD5:sfGFP fusion in E.coli, we have proven droplet formation in the E.coli cytosol.

Fig. 1: E.coli cells expressing a SPD5:sfGFP fusion verify droplet formation

To confirm that the constructs were were seeing were in fact dynamic droplets and not dead aggregates, we bleached several regions of interest with a 475 nm laser beam. Due to the dynamic nature of the droplets, flurescent should be recovered by diffusion of unbleached proteins

Fig.2 Recovery of fluorescence after photobleaching in SPD5:sfGFP expressing C321.deltaA cells.