The development of synthetic cells is of great interest to identify the minimal requirements for cellular processes and to enable a range of novel biomedical applications. This project aims to provide both fundamental and practical insights into designing synthetic cells that recapitulate essential cellular functions, such as sensing, self-growth, and biological reactions. For example, sensing is a central function in how living cells make decisions for synthesis and controlled release and encapsulation of molecules throughout vesicle membranes. Proteins at cell membranes play a critical role in these cellular functions to mediate sensing and intracellular cascade reactions. This project will demonstrate these functions by rationally designing the protein building blocks using recombinant protein technology and by engineering their assembled vesicle structure and properties to make them respond to external stimuli. Interdisciplinary collaboration in the fields of polymer physics, synthetic biology, and protein engineering will be conducted in this project, combined with outreach educational efforts to provide hands-on learning opportunities to underrepresented students at different levels in Gainesville, Florida, and Lincolnshire, Illinois. <br/><br/>The main objective of this project is to build minimal synthetic cells capable of sensing and responding to external signals, by engineering the compositions and physical properties of globular protein vesicles (GPVs) through the bottom-up approach. The vesicle membrane of a synthetic cell will be made from recombinant fusion proteins composed of sensor head proteins, leucine zippers, and elastin-like polypeptides. The specific objectives are 1) to engineer GPVs for sensing specific chemicals by tuning lateral association of sensor proteins, 2) to demonstrate the sensing-induced transmembrane signaling to activate transcription of reporters, and 3) to design programmable signal transduction and amplification cascades upon sensing. This collaborative project combines a thorough understanding of the self-assembly of polymeric building blocks, cell-free synthetic biology, and molecular bioengineering of protein-ligand interactions and enzyme specificity. Ultimately, this project envisions the high potential of the protein-assembled synthetic cells, which can manipulate diverse protein functions in minimal cell structure.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.