Automated Small Bioreactors Show Time-Based Molecular Responses to Gene Suppression in Pseudomonas putida

A critical observation window for gene silencing was identified between 7.8 and 26.7 hours, corresponding to 6.4-23.5 cumulative cell doublings.

• Pre-existing target proteins can mask the effects of early gene silencing in CRISPR interference experiments.
• Extended growth periods are needed to reduce the abundance of these proteins, but prolonged repression can lead to the emergence of mutant cells that escape silencing.
• The integration of a regulated CRISPRi system with an automated bioreactor allows for continuous exponential growth, enabling detailed mapping of gene silencing dynamics.
• Multi-omics profiling of the arginine biosynthesis pathway revealed that specific gene knockdowns led to diverse changes in metabolic processes.
• This approach provides a scalable method to achieve precise temporal control in phenotypic characterization, enhancing the reliability of functional genomics.

Simplified