Unraveling Quantitative Relationships between Intracellular Phase Separation and Gene Expression through Single-Cell Analysis

After the first observation of spontaneous occurrence of phase separation (PS) a living cell, the ubiquity of PS in a living system has been demonstrated. While our biophysical understanding of PS has drastically improved, its biological functions are much less known. It has been suggested that PS may regulate gene expression by selectively sequestering biomolecules into microcompartments formed by PS, and their concentrations inside the microcompartments are greatly elevated. Hence, if sequestered biomolecules are transcription factors or other molecules involving in gene expression, the increased concentrations are expected to enhance their downstream gene expression. However, past studies reported contradictory results. In order to elucidate quantitative relationships between PS and gene expression, we employed a single-cell fluorescence imaging methodology to establish quantitative correlations between phase separation and gene expression in E. coli. Specifically, we expressed a wide range of transcription factors (TFs) fused with a fluorescent protein (FP) and a PS-inducing motif (PSM) so that TFs undergo PS. By observing expression levels of another FP induced by a TF at the single-cell level, we can construct a dose-response curve between TF and FP in a high-throughput manner. By analyzing how PS occurrence affects a dose-response curve, we can quantitatively establish their correlations. By constructing dose-response curves with different PSMs with distinctive PS behaviors, the proposed screening method will provide a high-throughput way to characterize a variety of PSMs so that we can employ them as a new genetic part to control gene dynamics in a living cell.