Unbiased tracking of the progression of mRNA and protein synthesis in bulk and in liposome-confined reactions

Abstract

The compartmentalization of a cell-free gene expression system inside a self-assembled lipid vesicle is envisioned as the simplest chassis for the construction of a minimal cell. Although crucial for its realization, quantitative understanding of the dynamics of gene expression in bulk and liposome-confined reactions is scarce. Here, we used two orthogonal fluorescence labeling tools to report the amounts of mRNA and protein produced in a reconstituted biosynthesis system, simultaneously and in real-time. The Spinach RNA aptamer and its fluorogenic probe were used for mRNA detection. Applying this dual-reporter assay to the analysis of transcript and protein production inside lipid vesicles revealed that their levels are uncorrelated, most probably a consequence of the low copy-number of some components in liposome-confined reactions. We believe that the stochastic nature of gene expression should be appreciated as a design principle for the assembly of a minimal cell.

Keywords: RNA aptamers; artificial cells; gene expression; liposomes; self-assembly.