Simultaneous monitoring of transcription and translation in mammalian cell-free expression in bulk and in cell-sized droplets.

Synthetic Biology
Shue WangAllen P Liu

Abstract

Transcription and translation are two critical processes during eukaryotic gene expression that regulate cellular activities. The development of mammalian cell-free expression (CFE) systems provides a platform for studying these two critical processes in vitro for bottom-up synthetic biology applications such as construction of an artificial cell. Moreover, real-time monitoring of the dynamics of synthesized mRNA and protein is key to characterize and optimize gene circuits before implementing in living cells or in artificial cells. However, there are few tools for measurement of mRNA and protein dynamics in mammalian CFE systems. Here, we developed a locked nucleic acid (LNA) probe for monitoring transcription in a HeLa-based CFE system in real-time. By using this LNA probe in conjunction with a fluorescent reporter protein, we were able to simultaneously monitor mRNA and protein dynamics in bulk reactions and cell-sized single-emulsion droplets. We found rapid production of mRNA transcripts that decreased over time as protein production ensued in bulk reactions. Our results also showed that transcription in cell-sized droplets has different dynamics compared to the transcription in bulk reactions. The use of this LNA probe in...Continue Reading

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Citations

Mar 11, 2020·Annual Review of Biomedical Engineering·Vincent Noireaux, Allen P Liu
Dec 14, 2018·Natural Computing·Yiyu XiangBaojun Wang
May 1, 2019·Micromachines·Christoph EilenbergerMario Rothbauer
Apr 9, 2020·Frontiers in Bioengineering and Biotechnology·Nadanai LaohakunakornSebastian J Maerkl
May 1, 2019·ACS Synthetic Biology·Shue WangAllen P Liu
Jan 22, 2022·ACS Synthetic Biology·David T GonzalesT-Y Dora Tang

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