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A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Published on: June 16, 2016

An improved single cell ultrahigh throughput screening method based on in vitro compartmentalization.

Fuqiang Ma¹, Yuan Xie², Chen Huang¹

¹State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.

|March 4, 2014

Summary

This summary is machine-generated.

This study introduces a novel membrane-extrusion method for in vitro compartmentalization based fluorescence-activated cell sorting (IVC-FACS), enhancing enzyme screening accuracy and speed. The improved IVC-FACS system enables ultrahigh-throughput screening of enzyme activity, accelerating biocatalyst discovery.

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Area of Science:

Biotechnology
Enzyme Engineering
Molecular Biology

Background:

High-throughput screening is crucial for enzyme discovery and engineering.
In vitro compartmentalization based fluorescence-activated cell sorting (IVC-FACS) is a powerful ultrahigh-throughput screening tool for biocatalysts.
Current IVC-FACS methods suffer from limited accuracy due to non-uniform micro-reactors.

Purpose of the Study:

To develop an improved IVC-FACS protocol for more accurate and efficient ultrahigh-throughput screening of enzymatic activity.
To enhance the uniformity of micro-reactors for improved assay precision.
To demonstrate the utility of the improved system in enzyme evolution and directed evolution applications.

Main Methods:

A membrane-extrusion technique was employed to generate uniform micro-reactors.
The modified IVC-FACS protocol was used for ultrahigh-throughput screening of enzymatic activity (>10⁸ clones/day).
Directed evolution was performed on a thermophilic esterase (AFEST) using the new system.

Main Results:

The improved IVC-FACS system achieved high accuracy, discriminating two-fold differences in enzymatic activity.
Enzymatic reactions within micro-reactors exhibited bulk-like kinetics and a wide dynamic range.
E. coli cells with esterase activity were enriched 330-fold in a single sorting round.
Directed evolution of AFEST resulted in mutants with approximately two-fold higher catalytic activity, approaching diffusion-limited efficiency.

Conclusions:

The membrane-extrusion based IVC-FACS protocol significantly enhances the accuracy and throughput of biocatalyst screening.
This improved system facilitates the discovery and engineering of enzymes with superior properties.
The methodology is effective for directed evolution, leading to substantial improvements in enzyme performance.