A cloning vector for creation of Escherichia coli lacZ translational fusions and generation of linear template for chromosomal integration
- 作者：Gaylen A. Uhlich ; gaylen.uhlich@ars.usda.gov ; Chin-Yi Chen
- 关键词：STEC ; 尾-Galactosidase ; Promoter expression ; lacZ translational fusion
- 刊名：Plasmid
- 出版年：2012
- 期刊代码：124_0147619x
- 类别：abs
- 出版时间：May, 2012
- 卷：67
- 期：3
- 页码：259-263
- 文件大小：372 K
摘要
A novel cloning vector to aid in the construction of single copy 尾-galactosidase reporter systems for gene expression studies in lactose metabolizing Escherichia coli strains, including STEC, is described. The plasmid allows construction of translational fusions of cloned gene promoters to a short segment of E. coli lacZ. A selectable spectinomycin resistance marker flanked by a short lacI segment is positioned 5鈥?to the cloning site. PCR amplification using opposing primers complementary to the upstream lacI fragment and the downstream lacZ fragment generates a linear template suitable for integration using pRedET recombination. Integration of linear template derived from the recombinant plasmid into host strains replaces the entire native lacZ promoter and fuses the promoter of interest in-frame with the lacZ gene, thus simultaneously producing a single-copy, chromosomal reporter system and eliminating background lacZ expression. Studies comparing ahpC expression from a chromosomal fusion in the lac open with that on a plasmid in E. coli strain EDL933 are shown.