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.
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.
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Last modified October 14, 2003