Figure 1

Bacillus anthracis Targetron plasmid. (A) A variant of the Ll.LtrB group II intron designed to insert into orfB of IS605 elements of Bacillus anthracis was cloned into a Bacillus anthracis/E. coli. shuttle vector with a kanamycin resistance marker (bacillus) and ampicillin marker for E. coli. The intron and intron encoded LtrA protein are driven from a Cadmium inducible promoter (Cd). (B) EBS1/δ and EBS2 (shown schematically as black bars in the intron) are sequences within the intron controlling exon recognition via base pairing interactions. Reprogrammed sequences in EBS1/δ and EBS2 for efficient integration into IS605 are shown. (C) Plasmid IS605tt pRB373 was introduced into Bacillus anthracis Sterne via electroporation and selection for kanamycin resistant colonies. PCR screening of individual colonies using primers flanking the potential site of intron insertion show virtually all colonies had an intron insertion. There are 3 genomic copies of IS605 and in the two lanes highlighted by a white arrow all three copies have an intron insertion. Lanes marked M have 1 kb molecular weight markers.