New ultrarare restriction site-carrying transposons for bacterial genomics

Electrophoretic separation of macrorestriction fragments containing a particular genomic interval has until recently depended on fortuitously placed native rare restriction sites. We present new IS10-based transposons carrying the yeast intron-encoded I-SceI restriction site which is absent from most prokaryotic and eukaryotic genomes. Construction of the plasmid vectors containing them is described. Analysis by conventional or Pulsed Field gel electrophoresis of the DNA fragments generated by the I-SceI digestion reveals the physical distance between genomic insertions of these transposons: use of the same approach to subdivide the chromosome of Escherichia coli K-12 into equivalently sized contiguous/nonoverlapping I-SceI fragments is demonstrated. Because coordinates for the loci delimited by their insertions can be readily determined in different isolates by either physical or genetic manipulations, these transposons allow sufficient flexibility for species-wide bacterial genomics.