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Excision of Unstable Artificial Gene-Specific Inverted Repeats Mediates Scar-Free Gene Deletions in Escherichia coli

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Abstract

Inverted repeat and palindromic sequences have the propensity to form non-beta cruciform structures during DNA replication, leading to perturbations within the genome or plasmid replicon. In this study, the tolerance of the Escherichia coli genome to inverted repeat sequences from 25 to 1200 bp was investigated. Genomic inverted repeats were readily created via the homologous insertion of an overlap extension PCR product containing a gene-specific region of the genome together with thyA coding sequence, creating inverted repeat sequences of various lengths flanking the thyA selection marker in the resulting genome. Inverted repeat sequences below 100 bp were stably propagated, while those above and up to 1200 bp were found to be transiently unstable under auxotrophic thymine selection. Excision efficiency improves with increases of the inverted repeat until 600–800 bp, indicating that the genomic stability of inverted repeat sequences is due to secondary structure formation. Its effectiveness of creating precise and scar-free gene deletions was further demonstrated by deleting a number of genes in E. coli. The procedure can be readily adapted for sequence integration and point mutations in E. coli genome. It also has the potential for applications on other bacteria for efficient gene deletions.

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Acknowledgments

We thank the group members of Industrial Biotechnology, Institute of Chemical & Engineering Sciences, for their insights and discussion. This work was funded by A*STAR in Singapore ICES/12-574A01. We thank the reviewer for the comments and constructive criticisms.

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Authors and Affiliations

  1. Industrial Biotechnology Division, Institute of Chemical & Engineering Sciences, Agency for Science, Technology and Research (A*STAR), 1 Pesek Road, Jurong Island, Singapore, 627833

    Crystal Jing Ying Tear, Chanyuen Lim & Hua Zhao

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  1. Crystal Jing Ying Tear
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  2. Chanyuen Lim
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  3. Hua Zhao
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Correspondence to Hua Zhao.

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Crystal Jing Ying Tear and Chanyuen Lim contributed equally.

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Tear, C.J.Y., Lim, C. & Zhao, H. Excision of Unstable Artificial Gene-Specific Inverted Repeats Mediates Scar-Free Gene Deletions in Escherichia coli . Appl Biochem Biotechnol 175, 1858–1867 (2015). https://doi.org/10.1007/s12010-014-1402-4

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