| Abstract: | We describe the construction of a system allowing high-frequency transposition and deletion mutagenesis with class-II transposons containing a kanamycin or a chloramphenicol-resistance marker. The system utilizes the transposition function of Tn3 and the resolution function of Tn951/Tn2501 which leads to an uncoupling of the resolution and repression functions. It consists of defective transposons inserted into conjugative, replication thermosensitive plasmids. The properties of the system are: easily selectable resistance markers, high transposition frequencies onto plasmids, low transposition frequencies onto the host chromosome, placement of the tnpA gene outside the transposons so that "second-generation" transposition does not occur, possibility to transpose the whole system onto other plasmid vectors with different selection strategies, consecutive use of two transposons for deletion mutagenesis and restriction mapping. |
