Monitoring mRNA and protein levels in bulk and in model vesicle-based artificial cells.

Abstract

With rising interest in utilizing cell-free gene expression systems in bottom-up synthetic biology projects, novel labeling tools need to be developed to accurately report the dynamics and performance of the biosynthesis machinery operating in various reaction conditions. Monitoring the transcription activity has been simplified by the Spinach technology, an RNA aptamer that emits fluorescence upon binding to a small organic dye. Recently, we tracked the fluorescence of Spinach-tagged messenger RNA (mRNA) and its translation product the yellow fluorescent protein (YFP), both synthesized in the protein synthesis using recombinant elements system from a DNA template. Building on our previous study, we describe here an improved Spinach reporter with modified flanking sequences that confer higher propensity for aptamer folding and, thus, enhanced fluorescence brightness. Hence, the kinetics of mRNA and YFP production could be simultaneously monitored with unprecedented sensitivity. A combination of methodologies, comprising RNA gel analysis, real-time quantitative polymerase chain reaction, absorbance measurements, and fluorescence correlation spectroscopy, was used to convert fluorescence intensity units into absolute concentrations of transcript and YFP translational product. Furthermore, we demonstrated that the new Spinach construct greatly enhanced mRNA detection when gene expression was confined inside self-assembled lipid vesicles. Therefore, we argue that this assay could be used to evaluate systematically the performance of transcription and translation in model vesicle-based artificial cells.