Delivery of Transposons

Transposition typically occurs at a low frequency in vivo. Therefore, it is essential to have an efficient delivery system to isolate a collection of transposon insertions in a host. A good delivery system provides a selection for transposition. A variety of approaches are commonly used.

  1. Phage delivery systems. Phage delivery systems take advantage of a transposon insertion on a phage that is unable to lyse or lysogenize the recipient cell.

    For example, lambda cI::Tn10 P(Am) cannot form lysogens because the Tn10 insertion disrupts the cI gene, and cannot grow lytically in a supo recipient because the P gene product is required for phage replication. A lysate of this phage is prepared on an E. coli amber suppressor mutant, then an E. coli supo recipient is infected with the phage, selecting for tetracycline resistance (TetR) encoded by the Tn10. The resulting TetR colonies are due to transposition of Tn10 from the disabled phage onto the chromosome.

    An analogous approach relies on the transfer of phage carrying a transposon from one bacterial species where the phage can reproduce to a species where the phage can infect but cannot replicate. For example, phage P1 can efficiently infect and replicate in E. coli, and can infect Myxococcus xanthus but cannot replicate in M. xanthus. A lysate of P1 carrying a transposon is grown in E. coli, then this lysate is used to infect M. xanthus. When M. xanthus is infected with a lysate of P1::Tn5 with selection for kanamycin resistance (KanR) encoded by the Tn5, the resulting KanR colonies are due to transposition of Tn5 from the disabled phage onto the chromosome.

  2. Plasmid delivery systems. Plasmid delivery systems take advantage of a transposon insertion on a plasmid that is unable to replicate in the recipient cell (i.e. the transposon is carried on a suicide plasmid). The plasmid can be transferred from the permissive host to the nonpermissive host by conjugation, transformation, or electroporation, with selection for an antibiotic resistance encoded by the transposon.

  3. Overexpression of transposase in trans. The frequency of transposition can often be improved by increasing the concentration of transposase in the cell. For example, the transposase gene(s) can be cloned from a transposon into a vector that places their expression under the control of an easily regulated promoter, such as the tac promoter or the ara promoter. When the transposon is introduced into a cell producing high levels of transposase, the frequency of transposition is sufficiently high that it can often be detected without a direct selection.

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Last modified October 14, 2003