A multilayer microfluidic platform for the conduction of prolonged cell-free gene expression

Ardjan van der Linden, Pascal Pieters, Maaruthy Yelleswarapu, Zoe Swank, Wilhelm T.S. Huck, Sebastian Maerkl, Tom de Greef (Corresponding author)

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.
LanguageEnglish
Article numbere59655
Number of pages14
JournalJournal of Visualized Experiments (JoVE)
Volume152
StateAccepted/In press - 1 Oct 2019

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Lab-On-A-Chip Devices
Microfluidics
Transcription
Gene expression
Multilayers
Gene Expression
Synthetic Biology
Networks (circuits)
Gene Regulatory Networks
Complex networks
Reaction products
Byproducts
Software
Genes
Research Personnel
Hardware
Network protocols
Fabrication
In Vitro Techniques

Cite this

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title = "A multilayer microfluidic platform for the conduction of prolonged cell-free gene expression",
abstract = "The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.",
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A multilayer microfluidic platform for the conduction of prolonged cell-free gene expression. / van der Linden, Ardjan; Pieters, Pascal; Yelleswarapu, Maaruthy; Swank, Zoe; Huck, Wilhelm T.S.; Maerkl, Sebastian; de Greef, Tom (Corresponding author).

In: Journal of Visualized Experiments (JoVE), Vol. 152, e59655, 01.10.2019.

Research output: Contribution to journalArticleAcademicpeer-review

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