Selective Enrichment of Slow-Growing Bacteria in a Metabolism-Wide CRISPRi Library with a TIMER Protein

Beuter, Dominik and Gomes-Filho, José Vicente and Randau, Lennart and Díaz-Pascual, Francisco and Drescher, Knut and Link, Hannes. (2018) Selective Enrichment of Slow-Growing Bacteria in a Metabolism-Wide CRISPRi Library with a TIMER Protein. ACS Synthetic Biology, 7 (12). pp. 2775-2782.

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Construction of pooled genetic variant libraries has become very fast and versatile. The current limitation of this technique is to select cells with a desired phenotype from very large libraries. Especially cells with poor fitness and slow growth are difficult to select because they are rapidly outcompeted by fitter cells. Here, we demonstrate selective and high-throughput enrichment of slow-growing strains using a fluorescent TIMER protein and flow cytometry. As a proof of principle, we created a metabolism-wide CRISPR interference library for Escherichia coli and enriched targets that interfere with amino acid metabolism. After enrichment of slow-growing cells, the CRISPRi library consisted almost entirely of targets that block amino acid biosynthesis. These results provide general guidelines for how to enrich slow-growing strains from a large pool of genetic variants, with applications in genetic screens, metabolic engineering, and synthetic biology.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Infection Biology > Microbiology and Biophysics (Drescher)
UniBasel Contributors:Drescher, Knut
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:American Chemical Society
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:23 Jun 2021 15:04
Deposited On:23 Jun 2021 15:04

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