EcI5, a group IIB intron with high retrohoming frequency: DNA target site recognition and use in gene targeting

Mobility assays for wild-type and mutant introns. Donor plasmids (pACD2X for Ll.LtrB and pACD2-EcI5 or derivatives expressing mutant IEPs for EcI5) and recipient plasmids (pBRR3-ltrB for Ll.LtrB and pBRR3-EcI5 for EcI5) were transformed into E. coli HMS174(DE3). The cells were grown at 37°C to early log phase and incubated with 0 or 100 μM IPTG for 1 h. The introns carry a phage T7 promoter in DIV and integrate into a target site cloned in the recipient plasmid upstream of a promoterless tet^R gene, thereby activating that gene (see Fig. 2 and Materials and Methods). After induction, cells were plated on LB agar containing ampicillin or ampicillin plus tetracycline, and mobility efficiencies were calculated as the ratio of (Tet^R+Amp^R)/Amp^R colonies. The bar graphs show the mean for three determinations, with the error bar indicating the standard deviation.