Identifying genome-wide targets of osmotic stress tolerance in E. coli using CRISPR-mediated forward engineering

Biological responses to environmental stress can arise from multiple genetic solutions encoding tolerance mechanisms. In order to investigate the response to osmotic pressure in E. coli, we used an automated CRISPR editing workflow to generate a diverse cell population that included gene knockouts and different strength promoter substitution libraries targeting almost every gene in the genome. The resulting 25,000-variant pooled library was studied under a titrated salt challenge and the population dynamics were tracked using plasmid barcodes. By mapping 300,000 data points, we identified clear enrichment and depletion patterns and connected those back to functional genome annotations. We were able to rapidly validate our experiment with the performance of known salt stress tolerance loci, such as rpoE and osmE, and discover new gene targets.

During this webinar you will learn how to:

• Design experimental methods for studying osmotic stress response in E. coli
• Create genome-wide libraries of up 25,000 edits, including knockout and promoter substitutions, in an automated, massively parallel workflow
• Track diverging E. coli populations cultured in salt solutions at a range of concentrations
• Identify enriched and depleted variants, including genes known to be involved in the osmotic stress response and previously unknown targets

Who should attend?

• Core facility managers/lab managers (Directors, PIs, group leaders)
• Professors, research scientists, staff scientists, post-docs
• Research areas: Drug target identification, functional genomics, antimicrobial discovery and disease research, industrial bioproduction
• Technologies of interest: CRISPR, microbial gene editing, genome engineering, synthetic biology, forward engineering of microorganisms