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Direct Mutagenesis of Thousands of Genomic Targets Using Microarray-Derived Oligonucleotides
[Image: see text] 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...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical
Society
2014
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4304438/ https://www.ncbi.nlm.nih.gov/pubmed/24856730 http://dx.doi.org/10.1021/sb5001565 |
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author | Bonde, Mads T. Kosuri, Sriram Genee, Hans J. Sarup-Lytzen, Kira Church, George M. Sommer, Morten O.A. Wang, Harris H. |
author_facet | Bonde, Mads T. Kosuri, Sriram Genee, Hans J. Sarup-Lytzen, Kira Church, George M. Sommer, Morten O.A. Wang, Harris H. |
author_sort | Bonde, Mads T. |
collection | PubMed |
description | [Image: see text] 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. |
format | Online Article Text |
id | pubmed-4304438 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | American Chemical
Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-43044382015-01-27 Direct Mutagenesis of Thousands of Genomic Targets Using Microarray-Derived Oligonucleotides Bonde, Mads T. Kosuri, Sriram Genee, Hans J. Sarup-Lytzen, Kira Church, George M. Sommer, Morten O.A. Wang, Harris H. ACS Synth Biol [Image: see text] 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. American Chemical Society 2014-05-23 2015-01-16 /pmc/articles/PMC4304438/ /pubmed/24856730 http://dx.doi.org/10.1021/sb5001565 Text en Copyright © 2014 American Chemical Society This is an open access article published under an ACS AuthorChoice License (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) , which permits copying and redistribution of the article or any adaptations for non-commercial purposes. |
spellingShingle | Bonde, Mads T. Kosuri, Sriram Genee, Hans J. Sarup-Lytzen, Kira Church, George M. Sommer, Morten O.A. Wang, Harris H. Direct Mutagenesis of Thousands of Genomic Targets Using Microarray-Derived Oligonucleotides |
title | Direct Mutagenesis of Thousands of Genomic Targets
Using Microarray-Derived Oligonucleotides |
title_full | Direct Mutagenesis of Thousands of Genomic Targets
Using Microarray-Derived Oligonucleotides |
title_fullStr | Direct Mutagenesis of Thousands of Genomic Targets
Using Microarray-Derived Oligonucleotides |
title_full_unstemmed | Direct Mutagenesis of Thousands of Genomic Targets
Using Microarray-Derived Oligonucleotides |
title_short | Direct Mutagenesis of Thousands of Genomic Targets
Using Microarray-Derived Oligonucleotides |
title_sort | direct mutagenesis of thousands of genomic targets
using microarray-derived oligonucleotides |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4304438/ https://www.ncbi.nlm.nih.gov/pubmed/24856730 http://dx.doi.org/10.1021/sb5001565 |
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