Figure 2

Bacillus vector for selection of group II intron insertions. A schematic of the important vector features and selection scheme for group II intron based gene disruption. The plasmid is erythromycin resistant but kanamycin sensitve because a td group I intron interrupts its coding sequence in an antisense orientation. Transcription of the group II intron from the Ntr promoter allows self- splicing of the td intron and generates a substrate that is capable of genomic integration at pre-determined loci due to changes in sequences shown as black bars within the intron. Genomic integrants no longer have the td group I intron interrupting the kanamycin resistance gene and thus successful integrants are kanamycin resistant. For PCR verification of intron insertion, primers (shown as arrows above the genomic DNA schematic) were designed to flank the site of intron insertion in BAS4597 and BAS4553. kanamycin resistant colonies arising in Bacillus anthracis Sterne after transformation of plasmids reengineered to integrate the group II intron at BAS4597 and BAS4553 were screened by colony PCR and analyzed after electrophoresis on a 1% agarose gel in TBE buffer and visualized by staining with ethidium bromide. Lanes marked M have 1 Kb molecular weight markers.