a-multilayer-microfluidic-platform-for-conduction-prolonged-cell-free

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

bilayer-microfluidic-device-for-combinatorial-plug-production

Bilayer Microfluidic Device for Combinatorial Plug Production

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such ...

Radboud University

Eindhoven University of Technology

Liposomes are self-assembled phospholipid vesicles with great potential in fields ranging from targeted drug delivery to artificial cells. The formation of liposomes using microfluidic techniques has seen considerable progress, but the liposomes formation process itself has not been studied in great detail. As a result, high throughput, high-yielding routes to monodisperse liposomes with multiple compartments have not been demonstrated. Here, we report on a surfactant-assisted microfluidic route to uniform, single bilayer liposomes, ranging from 25 to 190 μm, and with or without multiple inner compartments. The key of our method is the precise control over the developing interfacial energies of complex W/O/W emulsion systems during liposome formation, which is achieved via an additional surfactant in the outer water phase. The liposomes consist of single bilayers, as demonstrated by nanopore formation experiments and confocal fluorescence microscopy, and they can act as compartments for cell-free gene expression. The microfluidic technique can be expanded to create liposomes with a multitude of coupled compartments, opening routes to networks of multistep microreactors.

Monodisperse Uni- and Multicompartment Liposomes.

Secondary structure formation of mRNA, caused by desynchronization of transcription and translation, is known to impact gene expression in vivo. Yet, inactivation of mRNA by secondary structures in cell-free protein expression is frequently overlooked. Transcription and translation rates are often not highly synchronized in cell-free expression systems, leading to a temporal mismatch between the processes and a drop in efficiency of protein production. By devising a cell-free gene expression platform in which transcriptional and translational elongation are successfully performed independently, we determine that sequence-dependent mRNA secondary structures are the main cause of mRNA inactivation in in vitro gene expression.

Protein Synthesis in Coupled and Uncoupled Cell-Free Prokaryotic Gene Expression Systems.

Vesosomes are nested liposomal structures with high potential as advanced drug delivery vehicles, bioreactors and artificial cells. However, to date no method has been reported to prepare monodisperse vesosomes of controlled size. Here we report on a multistep microfluidic strategy for hierarchically assembling uniform vesosomes from dewetting of double emulsion templates. The control afforded by our method is illustrated by the formation of concentric, pericentric and multicompartment liposomes. The microfluidic route to vesosomes offers an exceptional platform to build artificial cells as exemplified by the in vitro transcription in "nucleus" liposomes and the mimicry of the architecture of eukaryotic cells. Finally, we show the transport of small molecules across the nucleic envelope via insertion of nanopores into the bilayers.

Microfluidic Assembly of Monodisperse Vesosomes as Artificial Cell Models.

The compartmentalization of cell-free gene expression systems in liposomes provides an attractive route to the formation of protocells, but these models do not capture the physical (crowded) environment found in living systems. Here, we present a microfluidics-based route to produce monodisperse liposomes that can shrink almost 3 orders of magnitude without compromising their stability. We demonstrate that our strategy is compatible with cell-free gene expression and show increased protein production rates in crowded liposome protocells.

Macromolecularly Crowded Protocells from Reversibly Shrinking Monodisperse Liposomes.

Cell-free transcription-translation provides a simplified prototyping environment to rapidly design and study synthetic networks. Despite the presence of a well characterized toolbox of genetic elements, examples of genetic networks that exhibit complex temporal behavior are scarce. Here, we present a genetic oscillator implemented in an E. coli-based cell-free system under steady-state conditions using microfluidic flow reactors. The oscillator has an activator-repressor motif that utilizes the native transcriptional machinery of E. coli: the RNAP and its associated sigma factors. We optimized a kinetic model with experimental data using an evolutionary algorithm to quantify the key regulatory model parameters. The functional modulation of the RNAP was investigated by coupling two oscillators driven by competing sigma factors, allowing the modification of network properties by means of passive transcriptional regulation.

ArticleTotal : 2	Year
Publication title Cited by 16	2019
Publication title	2023

Article	Year
Monodisperse Uni- and Multicompartment Liposomes. Journal of the American Chemical Society\| PubMed ID: 27243596	2016
Protein Synthesis in Coupled and Uncoupled Cell-Free Prokaryotic Gene Expression Systems. ACS synthetic biology\| PubMed ID: 27306580	2016
Microfluidic Assembly of Monodisperse Vesosomes as Artificial Cell Models. Journal of the American Chemical Society\| PubMed ID: 27978623	2017
Macromolecularly Crowded Protocells from Reversibly Shrinking Monodisperse Liposomes. Journal of the American Chemical Society\| PubMed ID: 29870243	2018
Sigma Factor-Mediated Tuning of Bacterial Cell-Free Synthetic Genetic Oscillators. ACS synthetic biology\| PubMed ID: 30408412	2018

Maaruthy Yelleswarapu

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