An improved TnMax mini-transposon system suitable for sequencing, shuttle mutagenesis and gene fusions.

A new collection of mini-transposons (mini-Tn) of the previously described TnMax series [Haas et al, Gene 130 (1993a) 23-31] has been constructed. The transposons (Tn) bear genes conferring resistance to either chloramphenicol (Cm) or kanamycin (Km). Each member of the new series (TnMax5-TnMax11) contains the general M13 forward (M13-FP) and reverse (M13-RP1) sequencing primers close to the inverted repeats (IR), facilitating the rapid and convenient determination of the DNA sequences flanking the transposon insertion site. Furthermore, the mini-Tn possess the infrequently occurring NotI sites, allowing the localization of genes on macro-restriction maps of bacterial species. Some derivatives contain promoterless trp-lacZ (TnMax11), xylE (TnMax10), phoA (TnMax6) or blaM (TnMax7, TnMax9) genes next to the IR, suitable for the generation of in vivo gene- and operon fusions to study gene regulation, protein export, or to determine the topology of proteins in bacterial membranes. A set of conjugative minimal plasmid vectors (pMin1, pMin2) are used to select for TnMax insertions into the cloned insert, rather than the vector sequences. Due to the small size of the mini-Tn, and a simple and efficient mutagenesis procedure, the TnMax system is a useful tool for targeting and sequencing of cloned genes in Escherichia coli, and especially for shuttle mutagenesis of bacterial species which cannot be targeted by direct transposition.