Retargeting of EcI5 to insert into different sites in the E. coli lacZ gene. E. coli HMS174(DE3) was transformed with pACD3-EcI5 expressing the retargeted introns, grown at 37°C to early-log phase, and induced with 100 μM IPTG for 3 h unless noted otherwise. The cells were then plated on LB agar containing X-gal (40 mg/L), and the lacZ targeting frequency (Fig. 9) was determined by counting blue and white colonies. (A) Two-step PCR used to retarget the EcI5-ΔORF intron by modification of EBS and IBS sequences in the donor plasmid. The donor plasmid used as template was pACD3-EcI5A, -EcI5C, -EcI5G, or -EcI5T according to the desired EBS3 nucleotide residue. P1–P4 are primers used to modify the intron's EBS1 and EBS2 to be complementary to IBS1 and IBS2 in the DNA target site, and IBS1 and IBS2 in the 5′ exon of the donor plasmid to be complementary to the retargeted EBS1 and EBS2 for efficient RNA splicing (see Materials and Methods). The final PCR product containing the modified sequences was digested with XbaI and AvaII and swapped for the corresponding fragment of the same donor plasmid. (B) Colony PCR of E. coli lacZ disruptants obtained using retargeted EcI5 introns. Colony PCR was done with primers LacZP3 and LacZP4 flanking the intron-insertion site in the lacZ gene (see Supplemental Table 2). The figure shows representative data for three of the retargeted introns. “−colony” is a parallel PCR without a colony, and WT is a parallel PCR done on a colony of wild-type HMS174(DE3). (C) Southern hybridizations. Genomic DNA was isolated from E. coli HMS174(DE3) (WT) and the indicated lacZ disruptants grown under nonselective conditions. The DNA was digested with BglI, run in a 0.8% agarose gel, blotted to a nylon membrane, and hybridized with a 32P-labeled intron probe (see Materials and Methods). The donor plasmid pACD2-EcI5 digested with BglI was run in a parallel lane. The numbers to the left of the gel indicate the positions of size markers (1-kb plus ladder; Invitrogen). The schematics to the right depict the BglI fragments of the lacZ gene containing the inserted EcI5 intron. The insertion of targetron LacZ1257s, LacZ1790a, or LacZ1806s results in a 3.0-kb BglI fragment. The insertion of targetron LacZ163s disrupts the BglI sites at position 164, resulting in a larger fragment (4.7 kb). The LacZ1709a disruptant shows an additional band due to residual donor plasmid, which was retained in this disruptant but lost in the other disruptants during growth under nonselective conditions.
