Home › Cell Biology › Sealable Femtoliter Chamber Arrays for Cell-free Biology
Cell Biology JoVE (Open Access) Citable · DOI

Sealable Femtoliter Chamber Arrays for Cell-free Biology

DOI: 10.3791/52616-v
What you'll learn
  • ✓ Fabricate sealable femtoliter-volume PDMS microfluidic chambers using standard micromolding techniques
  • ✓ Load and seal cell-free protein synthesis reactants in microfluidic devices for gene expression studies
  • ✓ Acquire and analyze fluorescence data from confined cell-free reactions to measure stochastic gene expression
Protocol

A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene expression.

Difficulty
advanced
Total time
~3–5 days (including device fabrication, surface treatment, and reaction execution)
Biosafety
BSL-1

Steps

1
Fabricate PDMS microfluidic device with femtoliter chambers

Prepare polydimethylsiloxane (PDMS) devices using soft lithography and micromolding techniques to create sealable femtoliter-volume reaction chambers. This foundational step establishes the microfluidic architecture needed for cell-free reactions.

▶ 01:05
2
Set up microfluidic connections for protein synthesis

Connect the fabricated PDMS device to microfluidic tubing and establish fluid pathways for loading cell-free protein synthesis reactants. Ensure proper valve alignment and pressure regulation for controlled sample delivery.

▶ 05:48
3
Load and seal cell-free synthesis reactants

Load cell-free protein synthesis reaction mixtures into the femtoliter chambers and seal them to create confined reaction volumes. This encapsulation enables study of crowding and confinement effects on gene expression.

▶ 08:25
4
Acquire fluorescence microscopy data from reactions

Record time-lapse fluorescence images of sealed chambers during protein synthesis to capture real-time gene expression dynamics in confined volumes. Monitor stochastic variability across multiple individual reaction chambers.

▶ 08:25
5
Analyze images and process expression data

Extract fluorescence intensity traces from chamber images and quantify protein expression kinetics. Apply image analysis algorithms to measure stochastic gene expression processes at the single-reaction level.

▶ 11:00
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