Genetically encoded phospholipid production for autonomous synthetic cell proliferation

Cells, the building blocks of life, are vastly complex. This complexity confers to every living organism the ability to maintain oneself, reproduce oneself, and evolve. Creating a minimal system from nonliving components that is capable of self-maintenance, self-reproduction, and evolvability, will greatly increase our understanding of life. Essential features of every cell, synthetic or otherwise, are the compartment, a form of information transfer, and the ability to proliferate. In chapter 1, I argue that autonomy, that is self-governance, is another key characteristic of life. Therefore, to create a synthetic cell, aforementioned features should be recapitulated in an autonomous manner. This informs the synthetic cell design that is adhered to in this thesis. Phospholipid vesicles, so called liposomes, serve as a compartment. Reconstitution of the central dogma of molecular biology by cell-free gene expression with the PURE system enables the use of a genetic program encoded on DNA. By encoding its working instructions on its own DNA, the cell will be autonomous. Proliferation is carried out by a set of modules encoded on the DNA. These modules will serve to replicate the DNA itself, to replenish the gene expression machinery, and to stimulate growth and trigger division of the compartment. The goal of this thesis is to reconstitute compartment growth by cell-free gene expression of DNA encoding for phospholipid synthesis machinery.