Excision of Unstable Artificial Gene-Specific Inverted Repeats Mediates Scar-Free Gene Deletions in Escherichia coli
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- 作者：Crystal Jing Ying Tear ; Chanyuen Lim ; Hua Zhao
- 关键词：Inverted repeats ; Size dependent ; Scar ; free gene deletion ; E. coli
- 刊名：Applied Biochemistry and Biotechnology
- 出版年：2015
- 出版时间：February 2015
- 年：2015
- 卷：175
- 期：4
- 页码：1858-1867
- 全文大小：414 KB
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文摘
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 600-00?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.