期刊
ACS SYNTHETIC BIOLOGY
卷 4, 期 1, 页码 17-22出版社
AMER CHEMICAL SOC
DOI: 10.1021/sb5001565
关键词
genome engineering; MAGE; metabolic engineering; microarray; library synthesis
资金
- National Institutes of Health Director's Early Independence Award [1DP5OD009172-01]
- Department of Energy (DOE)
- National Science Foundation (NSF) [SA5283-11210]
- Novo Nordisk Foundation
- European Union Seventh Framework Programme [FP7-KBBE-2013-7-single-stage, 613745]
- Novo Nordisk Fonden [NNF10CC1016517] Funding Source: researchfish
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.
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