An assay to monitor the activity of DNAtransposition complexes yields a generalquality control measure for transpositionalrecombination reactions

摘要

Transposon-based technologies have many applications in molecular biology and can be used for gene delivery intoprokaryotic and eukaryotic cells. Common transpositional activity measurement assays suitable for many types oftransposons would be beneocial, 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 DNAtransposition complexes (transpososomes), using phage Mu transposition as a test platform. This assay quantioestranspositional recombination efociency and is based on an in vitro transposition reaction with a target plasmidcarrying a lethal ccdB gene. If transposition targets ccdB, this gene becomes inactivated, enabling plasmid-receivingEscherichia coli cells to survive and to be scored as colonies on selection plates. The assay was validated with 3 mini-Mutransposons varying in size and differing in their marker gene constitution. Tests with different amounts of transposonDNA provided a linear response and yielded a 10-fold operational range for the assay. The colony formation capacitywas linearly correlated with the competence status of the E.coli cells, enabling normalization of experimental dataobtained with different batches of recipient cells. The developed assay can now be used to directly comparetranspososome activities with all types of mini-Mu transposons, regardless of their aimed use. Furthermore, the assayshould be directly applicable to other transposition-based systems with a functional in vitro reaction, and it provides adependable quality control measure that previously has been lacking but is highly important for the evaluation ofcurrent and emerging transposon-based applications.