Molecular kinetics dictate population dynamics in CRISPR-based plasmid defense

Area of Science:

• Microbiology
• Systems Biology
• Synthetic Biology

Background:

• Mobile genetic elements, particularly plasmids, pose challenges in synthetic biology, agriculture, and medicine due to their complex spread dynamics.
• Understanding plasmid transmission requires integrating molecular kinetics, single-cell variability, and population structure.

Purpose of the Study:

• To characterize conjugative plasmid dynamics across molecular, single-cell, and population levels using advanced imaging techniques.
• To quantify the impact of CRISPR-Cas defense systems and plasmid features on plasmid spread.

Main Methods:

• Utilized time-lapse imaging to track fluorescently tagged conjugative plasmid RP4 and CRISPR-Cascade complexes.
• Employed single-cell tracking to measure conjugation rates, latent periods, and clearance kinetics.
• Developed a spatially resolved, agent-based model to predict population dynamics from single-cell parameters.

Main Results:

• Quantified population dynamics based on spacer target number, Cascade expression, and addiction modules.
• Reported conjugation rate per neighboring donor cell and estimated latent periods.
• Demonstrated successful prediction of plasmid population dynamics using single-cell biophysical parameters.

Conclusions:

• Plasmid dynamics result from a complex interplay between defense mechanisms, genetic elements, cell-cell interactions, and cost-benefit trade-offs.
• The developed imaging and modeling approach can disentangle factors governing community-wide plasmid dynamics.