Liquid–liquid phase separation (LLPS) of biological macromolecules is a biophysical principle that explains the formation of biomolecular condensates, also known as membraneless organelles, which enable intracellular compartmentalization within the cytoplasm and nucleus without the use of lipid membranes. Since this phenomenon was first reported in 2009, LLPS has been extensively investigated across diverse areas of molecular and cell biology.

In this laboratory course, students will investigate the physicochemical properties of phase-separating proteins through in silico analyses and experimentally examine their phase-separation behavior both in vitro and in cellulo. Through this series of integrated exercises, students will develop a fundamental understanding of biomolecular phase separation.

Week 1: Introduction
Weeks 2–7: Selection of a gene of interest (GOI), in silico analysis, cloning, PCR, plasmid preparation, sequencing, presentation, and discussion (Session 1)
Week 8: Midterm examination period (no laboratory class)
Weeks 9–10: In cellulo studies, optoDroplet assay, and stress granule induction
Week 11: Presentation and discussion (Session 2)
Weeks 12–14: In vitro studies, protein preparation, droplet assay, and phase-diagram construction
Week 15: Presentation and discussion (Session 3)
Week 16: Final examination period