Home › Cell Biology › A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression
Cell Biology JoVE (Open Access) Citable · DOI

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

DOI: 10.3791/59655-v
What you'll learn
  • ✓ Assemble and operate a multilayer PDMS microfluidic device for cell-free protein synthesis
  • ✓ Configure automated cooling and reagent delivery systems for prolonged IVTT reactions
  • ✓ Calibrate microfluidic hardware and interpret protein expression results in real time
Protocol

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

Difficulty
advanced
Total time
~4–6 hours (setup, calibration, and single experiment run)
Biosafety
BSL-1

Steps

1
Prepare off-chip cooling system components

Assemble and configure the external cooling setup required to maintain optimal temperature for prolonged cell-free gene expression reactions. This ensures stable conditions throughout the experiment.

▶ 01:00
2
Prepare experimental setup and calibrate equipment

Configure hardware, software interfaces, and peristaltic pumps to automate reagent delivery and maintain reaction parameters. Verify all connections before loading the microfluidic device.

▶ 02:35
3
Connect uncooled reagents to microfluidic device

Attach tubing and syringes containing room-temperature reagents to the microfluidic platform inlets. Ensure secure connections to prevent leakage during operation.

▶ 04:20
4
Connect cooled fluids to microfluidic device

Integrate temperature-controlled fluid lines from the off-chip cooling system to the device, maintaining consistent thermal conditions for sustained IVTT reactions.

▶ 05:45
5
Load reaction mixture into microfluidic device

Carefully prime and fill the multilayer device with cell-free transcription-translation extract and DNA template, avoiding air bubbles that could disrupt reaction kinetics.

▶ 06:49
6
Calibrate microfluidic device for optimal flow

Adjust pump rates and verify fluid flow uniformity across all channels using the software interface to ensure consistent reaction conditions.

▶ 08:09
7
Monitor deGFP protein expression results

Record fluorescence data as the device expresses enhanced green fluorescent protein over time, demonstrating sustained IVTT activity in the microfluidic platform.

▶ 08:56
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