FIGURE 5.

Genetic assay of LtrA-promoted splicing of the Ll.LtrB-ΔDIVa intron and colony-based immunoblot assay for GFP expression. (A) Map of plasmid pELG2-ΔDIVa used in the genetic assay. The plasmid contains an ltrB/GFP fusion cloned downstream from a T7lac promoter (arrow) and phage T7 φ10 gene Shine–Dalgarno (SD) sequence. The ltrB/GFP fusion is comprised of the first eight codons of the φ10 gene fused to a segment of the L. lactis ltrB gene consisting of the Ll.LtrB-ΔDIVa intron (open rectangle) and flanking exons (E1 and E2; filled boxes), with E2 linked in-frame to the GFP coding sequence (hatched). The LtrA ORF is cloned downstream from the GFP ORF. T1 and T2 are E. coli rrnB transcription terminators. The schematic below shows that splicing of the intron leads to the expression of GFP with a short N-terminal extension (42 amino acid residues) corresponding to the φ10 sequence and ligated exons fused to GFP. In the absence of splicing, translation is terminated by stop codons within the intron. (B) Colony-based immunoblot assay for GFP expression. E. coli HMS174(DE3) containing pELG2-ΔDIVa with and without ltrA (splicing competent and splicing defective, respectively) were mixed at a ratio of 1:10, then analyzed for GFP expression by colony immunoblot assay, as described in the Materials and Methods. The top shows an optical scan of the filter before treatment, and the bottom shows a chemiluminescence exposure of the filter after staining with anti-GFP antibody. Splicing-competent colonies producing GFP appear black on a light background.