Collaborative Research: Construction of DNA Programmed Minimal Cells with Membrane Mechanosensitive Functions

随着合成生物学的进步，现在有可能尝试建立一个成分最少的合成细胞，以便了解维持生命所需的基本元素是什么。在过去的十年里，已经提出了几种使用生命分子组装细胞类似物的方法。然而，到目前为止，还没有从零开始构建具有基本细胞功能的最小细胞，这一目标仍然是遥远的未来。特别是，构建细胞大小的具有活性感觉膜的遗传程序化脂囊还没有实现。这个项目的目标是构建一个最小的细胞，由一个封装在磷脂囊泡中的无细胞表达系统组成，能够通过表达机械敏感通道的脂双层来感知环境。这一步是合成细胞工程的核心，将提供必要的基本见解，以理解基本的生物学功能是如何产生的，这些功能表征了基因型和表型之间的联系。这项高度多学科的工作还将为如何设计真正具有功能的最小细胞系统提供见解，这些系统可能在其他研究领域有用，如生物技术和医学。所有级别的学生都将在跨学科的环境中接受培训。自下而上地构建复杂的体外生化系统是一个快速增长的研究领域。这项工作将为自下而上构建具有膜机械敏感功能的DNA编程最小细胞提供一条途径。本文的研究工作分为三个部分。将开发遗传编程来控制来自膜非渗透诱导剂的体外转录-翻译基因电路的激活。机械敏感通道，如来自大肠杆菌的MSCL，将被整合到脂质体的磷脂双层中，并将通过激活报告蛋白来测试其机械敏感性。最后，通过膜的机械激活将通过从化学前体合成脂质来耦合到双层的物理生长。这项工作解决了在细胞规模上整合分子部分以建立生物功能的基本问题。如果成功，这项工作将解决合成细胞大小的隔室的关键步骤，这些隔室具有与基因调控相结合的活性膜。

With advances in synthetic biology, it is now possible to try to build a synthetic cell with the minimum number of components (a minimal cell) in order to understand what are the essential elements needed to sustain life. Over the past decade, several approaches to assembling cell analogs using the molecules of life have been proposed. So far, however, no minimal cells capable of basic cell functions have been constructed from scratch, and this objective is still in a distant future. In particular, the construction of a cell-sized genetically programmed lipid vesicle with an active sensory membrane has not been achieved yet. The goal of this project is to construct a minimal cell, consisting of a cell-free expression system encapsulated into a phospholipids vesicle, capable of sensing the environment through its lipid bilayer by expressing mechanosensitive channels. This step, central for synthetic cell engineering, will provide fundamental insights necessary to understand how basic biological functions are created that characterize the link between genotype and phenotype. This highly multidisciplinary work will also provide insights on how to engineer truly functional minimal cell systems that could be useful in other research areas such as biotechnologies and medicine. Students at all levels will be trained in an interdisciplinary environment.The bottom-up construction of complex biochemical systems in vitro is a rapidly growing research area. The proposed work will offer a path towards bottom-up construction of DNA programmed minimal cells with membrane mechanosensitive functions. The research effort is divided into three parts. Genetic programming will be developed to control the activation of in vitro transcription-translation gene circuits from a membrane non-permeable inducer. Mechanosensitive channels such as MscL from E. coli will be incorporated into the phospholipids bilayer of liposomes and the mechanosensitivity will be tested by the activation of a reporter protein. Finally, mechanical activation through the membrane will be coupled to the physical growth of the bilayer by synthesizing lipids from chemical precursors. This work addresses fundamental questions about the integration of molecular parts at the scale of a cell to build biological functions. If successful, the work will address a critical step in the synthesis of cell-sized compartments with active membranes coupled to gene regulation.