Selective Enrichment of Slow-Growing Bacteria in a
Metabolism-Wide CRISPRi Library with a TIMER Protein
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Version 4 2018-11-18, 13:13
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Version 2 2018-11-16, 22:03
Version 1 2018-11-16, 18:06
Posted on 2018-11-19 - 11:03
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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Beuter, Dominik; Gomes-Filho, José Vicente; Randau, Lennart; Díaz-Pascual, Francisco; Drescher, Knut; Link, Hannes (2018). Selective Enrichment of Slow-Growing Bacteria in a
Metabolism-Wide CRISPRi Library with a TIMER Protein. ACS Publications. Collection. https://doi.org/10.1021/acssynbio.8b00379
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AUTHORS (6)
DB
Dominik Beuter
JG
José Vicente Gomes-Filho
LR
Lennart Randau
FD
Francisco Díaz-Pascual
KD
Knut Drescher
HL
Hannes Link