Bonde, Mads3; Kosuri, Sriram7; Genee, Hans Jasper3; Sarup-Lytzen, Kira1; Church, George M.7; Sommer, Morten3; Wang, Harris H.8
1 Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark2 Bacterial Cell Factories, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark3 Department of Systems Biology, Technical University of Denmark4 Drug Resistance and Community Dynamics, Department of Systems Biology, Technical University of Denmark5 Harvard University6 Columbia University7 Harvard University8 Columbia University
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 loci 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.
A C S Synthetic Biology, 2015, Vol 4, Issue 1, p. 17-22