An enhanced vector-free allele exchange (VFAE) mutagenesis protocol for genome editing in a wide range of bacterial species

文献类型： 外文期刊

第一作者： Gomaa, Ahmed E.

作者： Gomaa, Ahmed E.;Zhang, Chen;Yang, Zhimin;Shang, Liguo;Jiang, Shijie;Deng, Zhiping;Zhan, Yuhua;Lu, Wei;Lin, Min;Yan, Yongliang

作者机构：

关键词： Vector-free allele exchange (VFAE);Homologous recombination;Pseudomonas stutzeri;Escherichia coli;Bacillus subtilis;Genome editing

期刊名称：AMB EXPRESS （ 影响因子：3.298； 五年影响因子：3.427 ）

ISSN： 2191-0855

年卷期： 2017 年 7 卷

页码：

收录情况： SCI

摘要： 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 highfrequency 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 falsepositive 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.

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