Fast, Streamlined In Situ Genotyping of Bacterial Strains for Single-Cell and Subcellular Dynamics Profiling

Area of Science:

• Cell Biology
• Molecular Biology
• Biotechnology

Background:

• Temporal dynamics are crucial for cellular regulation but existing technologies face limitations in resolution, single-cell information, and throughput.
• Current methods like live-cell imaging combined with seqFISH struggle with probe aggregation and plasmid copy numbers that perturb cell physiology.

Purpose of the Study:

• To address technical challenges hindering the widespread adoption of live-cell imaging with in situ genotyping for studying dynamic cellular phenotypes.
• To develop an integrated and physiologically compatible solution for high-throughput single-cell and subcellular dynamics screening.

Main Methods:

• Implemented a surface-modification strategy to prevent fluorescent probe aggregation in microfluidic chips.
• Utilized barcoded copy-number-tunable plasmids to optimize live-cell imaging and post hoc genotyping.

Main Results:

• Achieved robust in situ genotyping overnight by suppressing probe aggregation.
• Preserved delicate subcellular dynamics and maintained genotyping sensitivity by decoupling imaging and genotyping requirements.

Conclusions:

• The developed methods offer a faster, minimally perturbative workflow for image-based bacterial screening.
• Expanded scalability and versatility of high-throughput screening for fundamental and synthetic biology research.