期刊
ACS SYNTHETIC BIOLOGY
卷 7, 期 12, 页码 2775-2782出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.8b00379
关键词
single-cell growth; CRISPR interference; flow cytometry; genome-scale metabolic model; amino acid biosynthesis
资金
- Deutsche Forschungsgemeinschaft [LI 1993/2-1, SFB 987]
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.
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