Chromosomal barcoding as a tool for multiplexed phenotypic characterization of laboratory evolved lineages

Area of Science:

• Microbiology
• Evolutionary Biology
• Biotechnology

Background:

• Adaptive laboratory evolution (ALE) is crucial for enhancing microbial tolerance and understanding resistance mechanisms.
• High-throughput ALE is powerful but bottlenecked by slow individual lineage characterization.
• Efficient phenotyping is essential for optimizing production strains and studying evolutionary dynamics.

Purpose of the Study:

• To develop a novel method for markerless genomic barcode insertion in Escherichia coli.
• To enable multiplexed phenotyping of evolved strains in pooled competition experiments.
• To streamline the characterization of lineages from adaptive evolution experiments.

Main Methods:

• Developed a markerless genetic barcode insertion system in E. coli using a dual-auxotrophic selection strategy.
• Created a barcoded E. coli library for pooled competition experiments.
• Applied the barcoded library to evolve antibiotic resistance and compared phenotyping results with conventional methods.

Main Results:

• Demonstrated successful markerless insertion of randomized genetic barcodes into E. coli.
• Showed that multiplexed phenotyping of barcoded strains correlates significantly with traditional susceptibility testing and growth rate measurements.
• Validated the efficiency of the barcoded library in adaptive evolution experiments for antibiotic resistance.

Conclusions:

• The novel barcoding method drastically reduces the workload for characterizing evolved microbial lineages.
• This approach enables efficient prioritization of strains for further in-depth analysis.
• Barcoded bacterial libraries offer new possibilities for community dynamics profiling and in vivo/in situ lineage tracking.