Gene transposition in bacteria will be studied by manipulation of the molecular components and reaction conditions of an in vitro transposition system using the Kanamycin-resistance transposon Tn5. Initially the efficiency and transpositional fidelty of the system must be optimized. When a consistently high level of in vitro transposition is achieved, the cell free lysate reaction system will be fractionated and reconstituted with purified components. The ultimate goal is to achieve in vitro transposition in a totally defined reaction. Transposition will also be studied in vivo with the goal of complementing the wealth of qualitative information about transposition with absolute kinetic measurements of gene transposition under various conditions. Also, transposition of Tn5 will be studied in avariety of different bacterial species using a broad host-range, plasmid vector with thermosensitive replication.
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