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

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.