title: An assay to monitor the activity of DNA transposition complexes yields a general quality control measure for transpositional recombination reactions.
abstract: Transposon-based technologies have many applications in molecular biology and can be used for gene delivery into prokaryotic and eukaryotic cells. Common transpositional activity measurement assays suitable for many types of transposons would be beneficial, as diverse transposon systems could be compared for their performance attributes. Therefore, we developed a general-purpose assay to enable and standardize the activity measurement for DNA transposition complexes (transpososomes), using phage Mu transposition as a test platform. This assay quantifies transpositional recombination efficiency and is based on anin vitrotransposition reaction with a target plasmid carrying a lethalccdBgene. If transposition targetsccdB, this gene becomes inactivated, enabling plasmid-receivingEscherichia colicells to survive and to be scored as colonies on selection plates. The assay was validated with 3 mini-Mu transposons varying in size and differing in their marker gene constitution. Tests with different amounts of transposon DNA provided a linear response and yielded a 10-fold operational range for the assay. The colony formation capacity was linearly correlated with the competence status of theE.colicells, enabling normalization of experimental data obtained with different batches of recipient cells. The developed assay can now be used to directly compare transpososome activities with all types of mini-Mu transposons, regardless of their aimed use. Furthermore, the assay should be directly applicable to other transposition-based systems with a functionalin vitroreaction, and it provides a dependable quality control measure that previously has been lacking but is highly important for the evaluation of current and emerging transposon-based applications.
authors.affiliation: Division of Genetics and Physiology; Department of Biology; University of Turku ; Turku, Finland.
authors.forename: Elsi
authors.initials: E
authors.lastname: Pulkkinen
authors.affiliation: Division of Genetics and Physiology; Department of Biology; University of Turku ; Turku, Finland.
authors.forename: Saija
authors.initials: S
authors.lastname: Haapa-Paananen
authors.affiliation: Division of Genetics and Physiology; Department of Biology; University of Turku ; Turku, Finland.
authors.forename: Harri
authors.initials: H
authors.lastname: Savilahti
keywords: ccdB gene; phage Mu; transposition activity measurement; transposon technology; transpososome