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

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Selective Enrichment of Slow-Growing Bacteria in a Metabolism-Wide CRISPRi Library with a TIMER Protein
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-11-13 00:00:00 , DOI: 10.1021/acssynbio.8b00379
Dominik Beuter ₁ , José Vicente Gomes-Filho ₁ , Lennart Randau ₁ , Francisco Díaz-Pascual ₁ , Knut Drescher _{1,

2} , Hannes Link ₁

Affiliation

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.

中文翻译：

使用TIMER蛋白在新陈代谢的CRISPRi文库中选择性富集缓慢生长的细菌

汇集的遗传变体文库的构建已变得非常快速和通用。该技术的当前局限性是从非常大的文库中选择具有所需表型的细胞。尤其是适应性差和生长缓慢的细胞很难选择，因为它们会很快被嵌合细胞所取代。在这里，我们展示了使用荧光TIMER蛋白和流式细胞仪对生长缓慢的菌株进行选择性和高通量富集。作为原理的证明，我们为大肠杆菌创建了一个新陈代谢的CRISPR干扰文库和富集的靶标会干扰氨基酸的代谢。富集缓慢生长的细胞后，CRISPRi文库几乎完全由阻断氨基酸生物合成的靶标组成。这些结果为如何从大量遗传变异中富集缓慢生长的菌株提供了一般指导，并应用于遗传筛选，代谢工程和合成生物学中。

更新日期：2018-11-13

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