TABLE 3.

ClosTron insertion frequencies with erythromycin or lincomycin selection

StrainTarget siteaFrequency of desired mutant among clones screenedb
%No. positive/no. screened
C. difficile 630Δerm CD3563226s1008/8
C. difficile 630Δerm sleC 493s00/20c
C. difficile 630Δerm sleC 128a1005/5
C. difficile R20291 sleC 128a1004/4
  • a Introns were inserted after the indicated number of bases from the start of the open reading frame in either the sense (s) or antisense (a) orientation.

  • b Genomic DNA was extracted from erythromycin-resistant (630Δerm) or lincomycin-resistant (R20291) clones at random and used in a PCR to amplify the intron-exon junction. One clone of each desired mutant was selected, and the intron insertion site was verified by sequencing.

  • c Further screening of a pool of genomic DNA from >100 erythromycin-resistant clones indicated that no desired mutants were present using the base 493 target site. The intron or target site was therefore judged to be inefficient (as occasionally occurs using group II intron technology), so another target (base 128 with the antisense orientation) was chosen for sleC.