Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation.

Area of Science:

• Biotechnology and Synthetic Biology
• Microfluidics and Single-Cell Analysis
• Bacterial Gene Expression Systems

Background:

• Recombinant protein production often relies on bulk measurements, overlooking cell-to-cell variations.
• Phenotypic heterogeneity in microbial populations can significantly reduce production yields, particularly for challenging proteins.
• Understanding single-cell behavior is crucial for optimizing recombinant protein production.

Purpose of the Study:

• To investigate and compare phenotypic heterogeneity in commonly used E. coli expression systems.
• To identify system-inherent specifications affecting growth and protein production at the single-cell level.
• To evaluate the impact of inducers, concentrations, and genetic modifications on expression homogeneity.

Main Methods:

• Utilized microfluidic single-cell analysis to dynamically monitor E. coli populations.
• Systematically analyzed PT7lac/LacI, PBAD/AraC, and Pm/XylS expression systems.
• Assessed variations in growth behavior, expression phenotypes, and inducer response.

Main Results:

• The widely used E. coli BL21(DE3) strain exhibited poor expression homogeneity and growth robustness.
• Inducer choice and the presence of inducer uptake systems critically influenced phenotypic heterogeneity.
• The modified lacY-deficient PT7lac/LacI and Pm/XylS systems demonstrated superior homogeneity and robustness.

Conclusions:

• Microfluidic analysis is essential for uncovering and addressing phenotypic heterogeneity in recombinant protein production.
• The modified PT7lac/LacI and Pm/XylS systems are recommended for precise and homogeneous bacterial gene expression in E. coli.
• Optimizing inducer strategies and genetic configurations is key to enhancing production consistency.