Medical and Biological Physics(DMBP)
Physique médicale et biologique (DPMB)

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Vincent NOIREAUX
Rockefeller University

From In Vitro Genetic Circuits to an Artificial Cell

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A cell-free expression extract has been used to assemble genetic circuits in vitro. The extract, which does not contained endogenous DNA and RNA, is used as a source of energy to carry out transcription and translation of plasmid genes. Expression stops after 6 hours due to energy consumption, the maximum protein production is two micromolar. In a ten microliters reaction volume, we engineered transcriptional activation and repression cascades, in which the protein product of each stage is the input required to drive or block the following stage. Substantial time delays and dramatic decreases in output production are incurred with each additional stage, due to a bottleneck at the translation machinery. Faster turnover of messengers RNA can relieve competition between genes and stabilize output against variations in input and parameters. To bring the system at the scale of the cell, the cytoplasmic extract has been encapsulated in phospholipids vesicles from one to a few tens of micrometers diameter. Accumulation of the GFP reporter protein shows that the expression is confined into the vesicles. A membrane pore has been expressed inside the vesicle to permeabilize the bilayer and allow a subsequent feeding of the vesicles with nutrients. With a continuous bioreactor, one can then develop functions to build a minimal cell.