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

随着合成生物学的进步，现在有可能尝试用最少数量的成分构建一个合成细胞（最小细胞），以便了解维持生命所需的基本元素是什么。在过去的十年中，已经提出了几种利用生命分子组装细胞类似物的方法。然而，到目前为止，还没有能够从头开始构建具有基本细胞功能的最小细胞，而且这一目标仍在遥远的未来。特别是，一个细胞大小的基因编程脂质囊泡与一个主动的感觉膜的结构尚未实现。该项目的目标是构建一个最小的细胞，包括一个包裹在磷脂囊泡中的无细胞表达系统，能够通过其脂质双分子层表达机械敏感通道来感知环境。这一步是合成细胞工程的核心，将为理解基因型和表型之间的联系特征的基本生物学功能是如何产生的提供必要的基本见解。这项高度跨学科的工作还将为如何设计真正功能最小的细胞系统提供见解，这些系统可能在生物技术和医学等其他研究领域有用。各个层次的学生都将在跨学科的环境中接受培训。体外复杂生化系统的自下而上构建是一个快速发展的研究领域。提出的工作将为具有膜机械敏感功能的DNA编程最小细胞的自下而上构建提供一条途径。研究工作分为三个部分。遗传编程将被开发来控制体外转录-翻译基因回路的激活从膜不渗透诱导剂。机械敏感通道，如来自大肠杆菌的msc将被纳入脂质体的磷脂双分子层，机械敏感性将通过报告蛋白的激活来测试。最后，通过膜的机械激活将通过化学前体合成脂质与双分子层的物理生长相结合。这项工作解决了关于在细胞尺度上整合分子部分以构建生物功能的基本问题。如果成功，这项工作将解决一个关键步骤，合成细胞大小的室与活性膜耦合基因调控。

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.