An enhanced vector-free allele exchange (VFAE) mutagenesis protocol for genome editing in a wide range of bacterial species
Abstract Vector-free allele exchange (VFAE) is a newly developed protocol for genome editing in Pseudomonas species. Although several parameters have been determined to optimize the procedures for obtaining a stable and high-frequency mutation, numerous false-positive clones still appear on the plat...
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doaj-88141b0fe73245d6bce580ca4571f6d22020-11-24T23:24:32ZengSpringerOpenAMB Express2191-08552017-06-01711710.1186/s13568-017-0425-yAn enhanced vector-free allele exchange (VFAE) mutagenesis protocol for genome editing in a wide range of bacterial speciesAhmed E. Gomaa0Chen Zhang1Zhimin Yang2Liguo Shang3Shijie Jiang4Zhiping Deng5Yuhua Zhan6Wei Lu7Min Lin8Yongliang Yan9Biotechnology Research Institute, Chinese Academy of Agricultural SciencesBiotechnology Research Institute, Chinese Academy of Agricultural SciencesBiotechnology Research Institute, Chinese Academy of Agricultural SciencesBiotechnology Research Institute, Chinese Academy of Agricultural SciencesBiotechnology Research Institute, Chinese Academy of Agricultural SciencesBiotechnology Research Institute, Chinese Academy of Agricultural SciencesBiotechnology Research Institute, Chinese Academy of Agricultural SciencesBiotechnology Research Institute, Chinese Academy of Agricultural SciencesBiotechnology Research Institute, Chinese Academy of Agricultural SciencesBiotechnology Research Institute, Chinese Academy of Agricultural SciencesAbstract Vector-free allele exchange (VFAE) is a newly developed protocol for genome editing in Pseudomonas species. Although several parameters have been determined to optimize the procedures for obtaining a stable and high-frequency mutation, numerous false-positive clones still appear on the plate, which increases the difficulty of finding the desired mutants. It has also not been established whether this protocol can be used for genome editing in other bacterial species. In the current study, the protocol was modified to dramatically decrease the occurrence of false-positive colonies using Pseudomonas stutzeri A1501 as a model strain. This improvement was reached by increasing the occurrence of circular-DNA cassettes of the correct size. Furthermore, the enhanced protocol was used to construct mutants in both the gram-negative Escherichia coli BL21 and gram-positive Bacillus subtilis 168 strains. The protocol works well in both strains, yielding ideal results with a low percentage of false-positive colonies. In summary, the enhanced VFAE mutagenesis protocol is a potential tool for use in bacterial genome editing.http://link.springer.com/article/10.1186/s13568-017-0425-yVector-free allele exchange (VFAE)Homologous recombinationPseudomonas stutzeriEscherichia coliBacillus subtilisGenome editing |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Ahmed E. Gomaa Chen Zhang Zhimin Yang Liguo Shang Shijie Jiang Zhiping Deng Yuhua Zhan Wei Lu Min Lin Yongliang Yan |
spellingShingle |
Ahmed E. Gomaa Chen Zhang Zhimin Yang Liguo Shang Shijie Jiang Zhiping Deng Yuhua Zhan Wei Lu Min Lin Yongliang Yan An enhanced vector-free allele exchange (VFAE) mutagenesis protocol for genome editing in a wide range of bacterial species AMB Express Vector-free allele exchange (VFAE) Homologous recombination Pseudomonas stutzeri Escherichia coli Bacillus subtilis Genome editing |
author_facet |
Ahmed E. Gomaa Chen Zhang Zhimin Yang Liguo Shang Shijie Jiang Zhiping Deng Yuhua Zhan Wei Lu Min Lin Yongliang Yan |
author_sort |
Ahmed E. Gomaa |
title |
An enhanced vector-free allele exchange (VFAE) mutagenesis protocol for genome editing in a wide range of bacterial species |
title_short |
An enhanced vector-free allele exchange (VFAE) mutagenesis protocol for genome editing in a wide range of bacterial species |
title_full |
An enhanced vector-free allele exchange (VFAE) mutagenesis protocol for genome editing in a wide range of bacterial species |
title_fullStr |
An enhanced vector-free allele exchange (VFAE) mutagenesis protocol for genome editing in a wide range of bacterial species |
title_full_unstemmed |
An enhanced vector-free allele exchange (VFAE) mutagenesis protocol for genome editing in a wide range of bacterial species |
title_sort |
enhanced vector-free allele exchange (vfae) mutagenesis protocol for genome editing in a wide range of bacterial species |
publisher |
SpringerOpen |
series |
AMB Express |
issn |
2191-0855 |
publishDate |
2017-06-01 |
description |
Abstract Vector-free allele exchange (VFAE) is a newly developed protocol for genome editing in Pseudomonas species. Although several parameters have been determined to optimize the procedures for obtaining a stable and high-frequency mutation, numerous false-positive clones still appear on the plate, which increases the difficulty of finding the desired mutants. It has also not been established whether this protocol can be used for genome editing in other bacterial species. In the current study, the protocol was modified to dramatically decrease the occurrence of false-positive colonies using Pseudomonas stutzeri A1501 as a model strain. This improvement was reached by increasing the occurrence of circular-DNA cassettes of the correct size. Furthermore, the enhanced protocol was used to construct mutants in both the gram-negative Escherichia coli BL21 and gram-positive Bacillus subtilis 168 strains. The protocol works well in both strains, yielding ideal results with a low percentage of false-positive colonies. In summary, the enhanced VFAE mutagenesis protocol is a potential tool for use in bacterial genome editing. |
topic |
Vector-free allele exchange (VFAE) Homologous recombination Pseudomonas stutzeri Escherichia coli Bacillus subtilis Genome editing |
url |
http://link.springer.com/article/10.1186/s13568-017-0425-y |
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