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.

Structure and Mechanism of RNA Mimics of Green Fluorescent Protein

“…In addition to expression in E. coli (46,50,54,63), several RNAs have been expressed as fusion RNAs and imaged in mammalian cells, including 5S, a small ncRNA transcribed by RNA polymerase III that associates with the large ribosomal subunit (46); 7SK, an ncRNA that associates with transcription complexes; and CGG-repeat RNAs, which are linked to fragile X-associated tremor and ataxia syndrome (FXTAS) (57). In each case, fluorescence was not detectable in cells expressing a control RNA, whereas fluorescence was detected in cells expressing the imaging tag (57).…”

Toward the assembly of a minimal divisome

“…Exchanging or diluting the external medium with the feeding solution leads to an osmotic pressure-driven membrane permeabilization, which temporarily enables selective uptake of all necessary nutrients and tRNAs, and initiates the internal synthesis of mRNA and proteins. The stochastic nature of the component entrapment and of the progression of transcription and translation could be evidenced and quantified van Nies et al 2013).…”

“…Bulk protein synthesis was performed as described previously (van Nies et al 2013), except that the PUREfrex reaction mixture was supplemented with BODIPY-LystRNA Lys (FluoroTect TM GreenLys, Promega), an in vitro translation labeling system. The sample was incubated for 3 h at 37°C, denatured for 2 min at 70°C and analyzed on a 12 % SDS-PAGE using a fluorescence gel imager (Typhoon, Amersham Biosciences).…”

“…Lipid vesicles were produced essentially as described previously (Nourian et al 2012;van Nies et al 2013), with some modifications. Briefly, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, Avanti Polar Lipids), N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TRITC-DHPE, Invitrogen), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG-biotin, Avanti Polar Lipids), all dissolved in chloroform, were mixed at a molar ratio DOPC:TRITC-DHPE:DSPE-PEG-biotin = 100:0.5:0.1 and added to 212-300-lm glass beads (SigmaAldrich) in a round bottom glass flask.…”

Controlled Transcription of Exogenous mRNA in Platelets Using Protocells

“…While active protocells have previously been injected into mice, [11] their ability to function within eukaryotic cells has not been conclusively demonstrated. Protocells typically contain coupled RNA and protein synthesis, but we focused on transcription to bypass the difficulties in co-encapsulating components of translation, [13] while maintaining a wide range of potential applications in gene and RNAi therapy. [14] …”