Fig 1.

Colony PCR results indicated that group II intron-based vectors efficiently targeted the C. beijerinckii 8052 pta (in the antisense direction) and buk (in the sense direction) genes. (A and B) Introns were inserted in the antisense and sense directions, respectively: PCR products with gene-specific primers from mutants would be 915 bp longer than those from the wild type, while in PCR with a gene-specific primer and an intron-specific primer amplified across gene-intron junctions in mutants, no product was amplified from the wild type (panels A and B are modified from the Sigma-Aldrich TargeTron user manual). (C) PCR with pta gene-specific primers produced a longer product from the mutant (lane 1) than from the wild type (lane 4); PCR with a gene-specific primer and an intron-specific primer obtained PCR products with mutants but not with the wild type (lane 2, PCR product from the mutant with pta-17/18a-F and EBS2 primers; lane 3, PCR product from the mutant with pta-17/18a-R and EBS universal primers; lane 5, control from the wild type for lane 2; lane 6, control from the wild type for lane 3). (D) PCR with buk gene-specific primers produced longer product from the mutant (lane 1) than from the wild type (lane 4); PCR with a gene-specific primer and an intron-specific primer obtained PCR products with mutants but not with the wild type (lane 2, PCR product from mutant with buk-532/533s-F and EBS universal primers; lane 3, PCR product from the mutant with buk-532/533s-R and EBS2 primers; lane 5, control from the wild type for lane 2; lane 6, control from the wild type for lane 3). Lane M, 100-bp DNA marker (New England BioLabs Inc., Ipswich, MA); the numbers on the left are the corresponding marker lengths, in bp.