Figure 2. Construction of LLPS-based compartment in E. coli.
A. Illustration of PhASE#1 phase module design.FUSLCD can phase separate in vivo , while GCN4 can further facilitate it. CIB1 is one of the two members in the light-responsive protein pair. See Figure 3A for further explanation.B. Illustration of the mechanism behind compartment construction. As its concentration rises beyond a threshold, the phase module will self-aggregate into membraneless compartments with a unique chemical environment. C, D. The process of compartment formation in E. coli. Most compartments nucleated around cell poles and could move around (C). Some aggregates emerged elsewhere, moved towards an end of the bacteria cell, and finally fused with other condensates (D). Scale bar, 1 μm. E. Homogeneous compartment formation in E. coli. Almost every bacterium contains compartments localized at their poles, when expressing PhASE#1 phase module. Phase module was under T7 promoter controlled by lacO . Either 0.25 mM or 1 mM IPTG was added to induce its expression. For more IPTG concentration gradients, please refer to Supplementary Figure 1. Scale bar, 2.5 μm. F. Fluidity of PhASE#1 phase modulein vivo. Fluorescence Recovery After Photo-bleaching (FRAP) was used to characterize the fluidity of the fusion protein. The recovery of fluorescence could be due to rapid protein exchange with the surrounding cytoplasm. The fluorescence signal of the highlighted condensate was normalized to that on the other side of the bacterium. Scale bar, 1 μm.