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Bacterial Expression Systems for Enzymatic Activity in Droplet-Based Microfluidics.
Christos S Karamitros1,2, Mickaël Morvan3, Aurélie Vigne3
1Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D37077 Goettingen, Germany.
Analytical Chemistry
|January 8, 2020
Summary
Periplasmic expression in E. coli is optimal for single-cell enzyme activity screening in microfluidics. This method enhances enzyme accessibility and yield for sensitive detection in functional screenings.
Area of Science:
- Biotechnology
- Microfluidics
- Molecular Biology
Background:
- Functional screenings in droplet-based microfluidics demand analysis of individual cell activities.
- Maintaining the genotype-phenotype link is crucial for enzyme screening, especially in directed evolution.
- Optimizing microbial expression systems is key for sensitive single-cell enzymatic activity detection.
Purpose of the Study:
- To compare three E. coli expression systems for l-asparaginase (l-ASNase) detection in microfluidics.
- To identify the most suitable system for sensitive, single-cell enzymatic activity screening.
- To evaluate periplasmic, cytoplasmic, and membrane-displayed expression of l-ASNase.
Main Methods:
- Development of three E. coli expression vectors for l-ASNase localization (cytoplasm, inner membrane, periplasm).
- Functional screening of enzymatic activity at the single-cell level using droplet-based microfluidics.
- Comparison of enzyme yield and substrate accessibility across different expression strategies.
Main Results:
- Periplasmic expression of l-ASNase demonstrated the highest efficiency.
- This strategy achieved a good balance between enzyme yield and substrate accessibility.
- Cell lysis was not required for detection, simplifying the screening process.
Conclusions:
- Periplasmic expression is the optimal strategy for sensitive l-asparaginase screening in microfluidic systems.
- This approach facilitates genotype-phenotype linkage for directed evolution and diversity screening.
- Periplasmic expression offers a robust platform for single-cell functional assays in microfluidics.
