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A versatile and highly efficient method for scarless genome editing in Escherichia coli and Salmonella enterica
BACKGROUND: Recently developed methods for genome editing in bacteria take advantage of the introduction of double-strand breaks by I-SceI in a mutation cassette to select for cells in which homologous recombination has healed the break and introduced a desired mutation. This elegantly designed meth...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4236582/ https://www.ncbi.nlm.nih.gov/pubmed/25255806 http://dx.doi.org/10.1186/1472-6750-14-84 |
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author | Kim, Juhan Webb, Anthony M Kershner, Jamie P Blaskowski, Stephen Copley, Shelley D |
author_facet | Kim, Juhan Webb, Anthony M Kershner, Jamie P Blaskowski, Stephen Copley, Shelley D |
author_sort | Kim, Juhan |
collection | PubMed |
description | BACKGROUND: Recently developed methods for genome editing in bacteria take advantage of the introduction of double-strand breaks by I-SceI in a mutation cassette to select for cells in which homologous recombination has healed the break and introduced a desired mutation. This elegantly designed method did not work well in our hands for most genes. RESULTS: We corrected a mutation in the gene encoding I-SceI that compromised the function of a previously used Red helper plasmid. Further, we found that transcription extending into the mutation cassette interferes with cleavage by I-SceI. Addition of two transcription terminators upstream of the cleavage site dramatically increases the efficiency of genome editing. We also developed an improved method for modification of essential genes. Inclusion of a segment of the essential gene consisting of synonymous codons restores an open reading frame when the mutation cassette is integrated into the genome and decreases the frequency of recombination events that fail to incorporate the desired mutation. The optimized protocol takes only 5 days and has been 100% successful for over 100 genomic modifications in our hands. CONCLUSIONS: The method we describe here is reliable and versatile, enabling various types of genome editing in Escherichia coli and Salmonella enterica by straightforward modifications of the mutation cassette. We provide detailed descriptions of the methods as well as designs for insertions, deletions, and introduction of point mutations. |
format | Online Article Text |
id | pubmed-4236582 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-42365822014-11-19 A versatile and highly efficient method for scarless genome editing in Escherichia coli and Salmonella enterica Kim, Juhan Webb, Anthony M Kershner, Jamie P Blaskowski, Stephen Copley, Shelley D BMC Biotechnol Methodology Article BACKGROUND: Recently developed methods for genome editing in bacteria take advantage of the introduction of double-strand breaks by I-SceI in a mutation cassette to select for cells in which homologous recombination has healed the break and introduced a desired mutation. This elegantly designed method did not work well in our hands for most genes. RESULTS: We corrected a mutation in the gene encoding I-SceI that compromised the function of a previously used Red helper plasmid. Further, we found that transcription extending into the mutation cassette interferes with cleavage by I-SceI. Addition of two transcription terminators upstream of the cleavage site dramatically increases the efficiency of genome editing. We also developed an improved method for modification of essential genes. Inclusion of a segment of the essential gene consisting of synonymous codons restores an open reading frame when the mutation cassette is integrated into the genome and decreases the frequency of recombination events that fail to incorporate the desired mutation. The optimized protocol takes only 5 days and has been 100% successful for over 100 genomic modifications in our hands. CONCLUSIONS: The method we describe here is reliable and versatile, enabling various types of genome editing in Escherichia coli and Salmonella enterica by straightforward modifications of the mutation cassette. We provide detailed descriptions of the methods as well as designs for insertions, deletions, and introduction of point mutations. BioMed Central 2014-09-25 /pmc/articles/PMC4236582/ /pubmed/25255806 http://dx.doi.org/10.1186/1472-6750-14-84 Text en Copyright © 2014 Kim et al.; licensee BioMed Central Ltd. 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 work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Methodology Article Kim, Juhan Webb, Anthony M Kershner, Jamie P Blaskowski, Stephen Copley, Shelley D A versatile and highly efficient method for scarless genome editing in Escherichia coli and Salmonella enterica |
title | A versatile and highly efficient method for scarless genome editing in Escherichia coli and Salmonella enterica |
title_full | A versatile and highly efficient method for scarless genome editing in Escherichia coli and Salmonella enterica |
title_fullStr | A versatile and highly efficient method for scarless genome editing in Escherichia coli and Salmonella enterica |
title_full_unstemmed | A versatile and highly efficient method for scarless genome editing in Escherichia coli and Salmonella enterica |
title_short | A versatile and highly efficient method for scarless genome editing in Escherichia coli and Salmonella enterica |
title_sort | versatile and highly efficient method for scarless genome editing in escherichia coli and salmonella enterica |
topic | Methodology Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4236582/ https://www.ncbi.nlm.nih.gov/pubmed/25255806 http://dx.doi.org/10.1186/1472-6750-14-84 |
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