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 descr ibed TnMax series [Haas et al., Gene 130 (1993a) 23-31] has been const ructed, The transposons (Tn) bear genes conferring resistance to eithe r chloramphenicol (Cm) or kanamycin (Km). Each member of the new serie s (TnMax5-TnMax11) contains the general M13 forward (M13-FP) and rever se (M13-RP1) sequencing primers close to the inverted repeats (IR), fa cilitating the rapid and convenient determination of the DNA sequences flanking the transposon insertion site. Furthermore, the mini-Tn poss ess the infrequently occurring NotI sites, allowing the localization o f genes on macro-resriction maps of bacterial species. Some derivative s contain promoterless trp-lacZ (TnMax11), xylE (TnMax10), phoA (TnMax 6) or blaM (TnMax7, TnMax9) genes next to the IR, suitable for the gen eration 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, rathe r 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 u seful tool for targeting and sequencing of cloned genes in Escherichia coli, and especially for shuttle mutagenesis of bacterial species whi ch cannot be targeted by direct transposition.