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ACS Synth Biol. 2015 Jan 16;4(1):17-22. doi: 10.1021/sb5001565. Epub 2014 Jun 20.

Direct mutagenesis of thousands of genomic targets using microarray-derived oligonucleotides.

Author information

1
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , DK-2970 Hørsholm, Denmark.

Abstract

Multiplex Automated Genome Engineering (MAGE) allows simultaneous mutagenesis of multiple target sites in bacterial genomes using short oligonucleotides. However, large-scale mutagenesis requires hundreds to thousands of unique oligos, which are costly to synthesize and impossible to scale-up by traditional phosphoramidite column-based approaches. Here, we describe a novel method to amplify oligos from microarray chips for direct use in MAGE to perturb thousands of genomic sites simultaneously. We demonstrated the feasibility of large-scale mutagenesis by inserting T7 promoters upstream of 2585 operons in E. coli using this method, which we call Microarray-Oligonucleotide (MO)-MAGE. The resulting mutant library was characterized by high-throughput sequencing to show that all attempted insertions were estimated to have occurred at an average frequency of 0.02% per locus with 0.4 average insertions per cell. MO-MAGE enables cost-effective large-scale targeted genome engineering that should be useful for a variety of applications in synthetic biology and metabolic engineering.

KEYWORDS:

MAGE; genome engineering; library synthesis; metabolic engineering; microarray

PMID:
24856730
PMCID:
PMC4304438
DOI:
10.1021/sb5001565
[Indexed for MEDLINE]
Free PMC Article
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