Lineage Tracking for Probing Heritable Phenotypes at Single-Cell Resolution.

Area of Science:

• Microbiology
• Evolutionary Biology
• Biotechnology

Background:

• Understanding microbial evolution requires simultaneous analysis of phenotype and genotype at the single-cell level.
• Current DNA sequencing offers high-resolution genotypic analysis, but robust single-cell phenotypic analysis methods are lacking.

Purpose of the Study:

• To develop and validate a drop-based millifluidic system for detecting heritable phenotypic changes in evolving bacterial populations.
• To demonstrate the system's utility in tracking phenotypic diversity during prolonged starvation conditions.

Main Methods:

• Individual bacterial cells were compartmentalized into 100 nL drops within a millifluidic system.
• Cellular growth and phenotypic changes were monitored over multiple generations using a fluorescent protein reporter.
• Heritable phenotypic variations were amplified through multi-generational growth, followed by DNA sequencing to identify corresponding mutations.

Main Results:

• The millifluidic system successfully detected heritable phenotypic changes in evolving bacterial populations.
• Phenotypic diversity rapidly increased in Escherichia coli populations during 30 days of starvation, with the emergence of distinct heritable phenotypes.
• Specific mutations linked to observed phenotypic classes were identified via DNA sequencing.

Conclusions:

• This scalable lineage-tracking technology provides a powerful tool for high-resolution, large-scale single-cell phenotyping.
• The approach has significant implications for microbiology, microbial population biology, and the study of evolutionary processes.