Beuter Dominik, Gomes-Filho José Vicente, Randau Lennart, Díaz-Pascual Francisco, Drescher Knut, Link Hannes
Max Planck Institute for Terrestrial Microbiology , 35043 Marburg , Germany.
Department of Physics , Philipps-Universität Marburg , 35032 Marburg , Germany.
ACS Synth Biol. 2018 Dec 21;7(12):2775-2782. doi: 10.1021/acssynbio.8b00379. Epub 2018 Nov 16.
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.
汇集遗传变异文库的构建已经变得非常快速且通用。该技术目前的局限性在于从非常大的文库中筛选出具有所需表型的细胞。特别是那些适应性差且生长缓慢的细胞很难被筛选出来，因为它们会迅速被适应性更强的细胞淘汰。在这里，我们展示了使用荧光TIMER蛋白和流式细胞术对生长缓慢的菌株进行选择性和高通量富集。作为原理验证，我们为大肠杆菌创建了一个全代谢范围的CRISPR干扰文库，并富集了干扰氨基酸代谢的靶点。在富集生长缓慢的细胞后，CRISPRi文库几乎完全由阻断氨基酸生物合成的靶点组成。这些结果为如何从大量遗传变异中富集生长缓慢的菌株提供了通用指导方针，可应用于基因筛选、代谢工程和合成生物学。
