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An electroporation-free method based on Red recombineering for markerless deletion and genomic replacement in the Escherichia coli DH1 genome
The λ-Red recombination system is a popular method for gene editing. However, its applications are limited due to restricted electroporation of DNA fragments. Here, we present an electroporation-free λ-Red recombination method in which target DNA fragments are excised by I-CreI endonuclease in vivo...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655456/ https://www.ncbi.nlm.nih.gov/pubmed/29065183 http://dx.doi.org/10.1371/journal.pone.0186891 |
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author | Wei, Yanlong Deng, Pingping Mohsin, Ali Yang, Yan Zhou, Huayan Guo, Meijin Fang, Hongqing |
author_facet | Wei, Yanlong Deng, Pingping Mohsin, Ali Yang, Yan Zhou, Huayan Guo, Meijin Fang, Hongqing |
author_sort | Wei, Yanlong |
collection | PubMed |
description | The λ-Red recombination system is a popular method for gene editing. However, its applications are limited due to restricted electroporation of DNA fragments. Here, we present an electroporation-free λ-Red recombination method in which target DNA fragments are excised by I-CreI endonuclease in vivo from the landing pad plasmid. Subsequently, the I-SceI endonuclease-cutting chromosome and DNA double-strand break repair were required. Markerless deletion and genomic replacement were successfully accomplished by this novel approach. Eight nonessential regions of 2.4–104.4 kb in the Escherichia coli DH1 genome were deleted separately with selection efficiencies of 5.3–100%. Additionally, the recombination efficiencies were 2.5–45%, representing an order of magnitude improvement over the electroporation method. For example, for genomic replacement, lycopene expression flux (3.5 kb) was efficiently and precisely integrated into the chromosome, accompanied by replacement of nonessential regions separately into four differently oriented loci. The lycopene production level varied approximately by 5- and 10-fold, corresponding to the integrated position and expression direction, respectively, in the E. coli chromosome. |
format | Online Article Text |
id | pubmed-5655456 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-56554562017-11-09 An electroporation-free method based on Red recombineering for markerless deletion and genomic replacement in the Escherichia coli DH1 genome Wei, Yanlong Deng, Pingping Mohsin, Ali Yang, Yan Zhou, Huayan Guo, Meijin Fang, Hongqing PLoS One Research Article The λ-Red recombination system is a popular method for gene editing. However, its applications are limited due to restricted electroporation of DNA fragments. Here, we present an electroporation-free λ-Red recombination method in which target DNA fragments are excised by I-CreI endonuclease in vivo from the landing pad plasmid. Subsequently, the I-SceI endonuclease-cutting chromosome and DNA double-strand break repair were required. Markerless deletion and genomic replacement were successfully accomplished by this novel approach. Eight nonessential regions of 2.4–104.4 kb in the Escherichia coli DH1 genome were deleted separately with selection efficiencies of 5.3–100%. Additionally, the recombination efficiencies were 2.5–45%, representing an order of magnitude improvement over the electroporation method. For example, for genomic replacement, lycopene expression flux (3.5 kb) was efficiently and precisely integrated into the chromosome, accompanied by replacement of nonessential regions separately into four differently oriented loci. The lycopene production level varied approximately by 5- and 10-fold, corresponding to the integrated position and expression direction, respectively, in the E. coli chromosome. Public Library of Science 2017-10-24 /pmc/articles/PMC5655456/ /pubmed/29065183 http://dx.doi.org/10.1371/journal.pone.0186891 Text en © 2017 Wei et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Wei, Yanlong Deng, Pingping Mohsin, Ali Yang, Yan Zhou, Huayan Guo, Meijin Fang, Hongqing An electroporation-free method based on Red recombineering for markerless deletion and genomic replacement in the Escherichia coli DH1 genome |
title | An electroporation-free method based on Red recombineering for markerless deletion and genomic replacement in the Escherichia coli DH1 genome |
title_full | An electroporation-free method based on Red recombineering for markerless deletion and genomic replacement in the Escherichia coli DH1 genome |
title_fullStr | An electroporation-free method based on Red recombineering for markerless deletion and genomic replacement in the Escherichia coli DH1 genome |
title_full_unstemmed | An electroporation-free method based on Red recombineering for markerless deletion and genomic replacement in the Escherichia coli DH1 genome |
title_short | An electroporation-free method based on Red recombineering for markerless deletion and genomic replacement in the Escherichia coli DH1 genome |
title_sort | electroporation-free method based on red recombineering for markerless deletion and genomic replacement in the escherichia coli dh1 genome |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655456/ https://www.ncbi.nlm.nih.gov/pubmed/29065183 http://dx.doi.org/10.1371/journal.pone.0186891 |
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