Figure 9. The BPP-1 DGR can be engineered to target a kanamycin-resistance gene.

(A) Donor and recipient constructs used for Kan^R gene targeting. The left panel shows two donor plasmids, pMX-Km1 and pMX-Km2, both with engineered TRs containing the last 36 bp of the Kan^R ORF. The Kan^R reporter gene on recipient VRs has a truncation of the last 6 codons and is placed upstream of the IMH and hairpin/cruciform elements. The recipient cassette was inserted between attL and bbp1 of phage BPP-1ΔATR* (Recipient Phage, middle) or in a pMMB208-derived plasmid (Recipient Plasmid, left). (B) Engineered BPP-1 DGRs can target a Kan^R reporter gene on a replicating phage. Targeting assays were carried out by phage BPP-1ΔATR*Kan^S lytic infection of RB50 cells carrying the indicated donor plasmids. Targeting efficiencies were determined as the relative numbers of Kan^R cells in lysogens generated with fresh RB50 cells and the progeny phages. The bars for Km1 and Km2 represent mean ± s.d. *P<0.01 in a Student's t test. (C) Engineered BPP-1 DGRs can target a Kan^R reporter gene on a prophage in the bacterial chromosome. BPP-1ΔATR*Kan^S lysogens transformed with indicated donor plasmids were used for targeting assays. Resulting cells were directly plated on selective (+Kan) or non-selective plates to determine relative numbers of Kan^R cells. The bars represent mean ± s.d. *P<0.05 in a Student's t test. (D) Engineered BPP-1 DGRs can target a Kan^R reporter gene on a plasmid. Following induction for donor plasmid expression and Kan^R gene targeting, RB50 cells transformed with both donor and recipient plasmids were plated on selective (+Kan) or non-selective plates to determine targeting efficiencies as in (C). The bars represent mean ± s.d. *P<0.002 in a Student's t test. ^†No Kan^R colonies were observed for RT-deficient donors and the numbers represent limits of detection.