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Submitted to: Plasmid Journal
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 12/9/2011 Publication Date: 5/1/2012 Citation: Uhlich, G.A., Chen, C. 2012. A cloning vector for creation of Escherichia coli lacZ translational fusions and generation of linear template for chromosomal integrations. Plasmid Journal. 67(3):259-263. Interpretive Summary: Bacterial analysis at the gene and protein level requires the use of special molecular entities that can be introduced into individual cells and can carry cloned DNA fragments. Such vehicles have been constructed to express foreign proteins for use as cell markers, facilitate recombination of cloned fragments into the host cells to delete or alter existing genes, and express recombinant proteins. Non-pathogenic, laoratory-adapted strains of Escherichia coli and the plasmids engineered for specific use in those strains have served as the cornerstone of bacterial molecular genetics. However, there are additional barriers in non-laboratory strains that have complicated the use of many of these molecular entity methods. In this study, we describe the construction of slowing plasmid that enables users, in one simple cloning step, to generate fusions to an unique enzyme. Enzyme production can then be measured to estimate the activity of specific gene-of-interest. This new plasmid entity provides researchers with a fast and simple tool to test and compare gene activity, and eliminates the barriers asociated with expression studies in certain E. coli strains. Technical Abstract: A novel cloning vector to aid in the construction of ß-galactosidase reporter systems for gene expression studies in lactose metabolizing strains of Shiga toxin producing Escherichia coli is described. The plasmid allows construction of translational fusions of cloned gene promoters with a short segment of E. coli lacZ. A selectable spectinomycin resistance marker flanked by a short lacI segment is positioned 5’ to the cloning site. PCR amplification using opposing primers complimentary 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 9th codon of the native lacZ gene, thus simultaneously producing a single copy, chromosomal expression system and eliminating background lacZ expression. Studies comparing ahpC expression from a promoter fused in the lac open with that of a plasmid fusion in E. coli strain EDL933 are shown. |
