title: 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.
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Lorentzweg 1, 2628 CJ, Delft (The Netherlands).
authors.forename: Pauline
authors.initials: P
authors.lastname: van Nies
authors.forename: Zohreh
authors.initials: Z
authors.lastname: Nourian
authors.forename: Maurits
authors.initials: M
authors.lastname: Kok
authors.forename: Roeland
authors.initials: R
authors.lastname: van Wijk
authors.forename: Jonne
authors.initials: J
authors.lastname: Moeskops
authors.forename: Ilja
authors.initials: I
authors.lastname: Westerlaken
authors.forename: Jos M
authors.initials: JM
authors.lastname: Poolman
authors.forename: Rienk
authors.initials: R
authors.lastname: Eelkema
authors.forename: Jan H
authors.initials: JH
authors.lastname: van Esch
authors.forename: Yutetsu
authors.initials: Y
authors.lastname: Kuruma
authors.forename: Takuya
authors.initials: T
authors.lastname: Ueda
authors.forename: Christophe
authors.initials: C
authors.lastname: Danelon
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D015374:Biosensing Techniques / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D008081:Liposomes / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics; D008958:Models, Molecular; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013269:Stochastic Processes
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D008081:Liposomes; D008164:Luminescent Proteins; D011506:Proteins; D012333:RNA, Messenger; C054562:yellow fluorescent protein, Bacteria
keywords: RNA aptamers; artificial cells; gene expression; liposomes; self-assembly