Collaborative Research: Designing Minimal Synthetic Cells Capable of Sensing and Self-Manipulation via Tunable Self-Assembly

合成细胞的发展对确定细胞过程的最低要求和使一系列新的生物医学应用成为可能是非常有意义的。该项目旨在为设计概括基本细胞功能的合成细胞提供基本和实用的见解，如感知、自我生长和生物反应。例如，感知是活细胞如何在整个囊泡膜中决定分子的合成、受控释放和封装的核心功能。细胞膜上的蛋白质在这些细胞功能中起着关键的作用，介导感觉和细胞内的级联反应。该项目将通过使用重组蛋白质技术合理设计蛋白质构建块，并通过设计其组装的囊泡结构和性质使其对外部刺激做出反应来展示这些功能。该项目将在聚合物物理、合成生物学和蛋白质工程领域进行跨学科合作，并结合外展教育努力，为佛罗里达州盖恩斯维尔和伊利诺伊州林肯郡不同级别的未被充分代表的学生提供实践学习机会。该项目的主要目标是通过自下而上的方法设计球状蛋白囊泡(GPV)的组成和物理性质，从而建立能够感知和响应外部信号的最小合成细胞。合成细胞的囊泡膜将由传感器头部蛋白、亮氨酸拉链和弹性蛋白样多肽组成的重组融合蛋白制成。具体目标是1)通过调节传感器蛋白的横向结合来设计GPV来感知特定的化学物质，2)展示传感诱导的跨膜信号以激活报告基因的转录，3)设计可编程的信号转导和扩增级联反应。这个合作项目结合了对聚合物构建块自组装、无细胞合成生物学以及蛋白质-配体相互作用和酶特异性的分子生物工程的透彻了解。最终，该项目展望了蛋白质组装合成细胞的高潜力，它可以在最小的细胞结构中操纵不同的蛋白质功能。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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. 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.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.