Mini-Tn7 Insertion in Bacteria With Multiple glmS-Linked attTn7 Sites: Example Burkholderia Mallei ATCC 23344

摘要

We recently constructed a series of broad-range mini-Tn7 vectors to address some of the problems inherent to plasmid-based cloning systems such as multi-copy state, necessity for continued selection and scarcity for many bacteria. The potential of the mini-Tn7 vectors for finding wide-spread biomedical and environmental applications, particularly for analysis of complex systems such as animal and biofilm models was demonstrated. In the presence of a helper plasmid encoding the site-specific transposition pathway, we showed that, with few exceptions, Tn7 insertions occured site- and orientation specifically at attTn7 sites downstream of bacterial glmS genes, with the number of possible insertions per genome roughly corresponding to the number of glmS genes present in these genomes. Our mini-Tn7 system is characterized by its versatility, simplicity, ease of use and ready adaptation to many bacteria. Here, we present protocols for use of our mini-Tn7 system in bacteria with single, multiple and secondary attTn7 sites.