Fig. 2

Overview of bacterial selection system and library design. a Sketch of a bacterium containing plasmids used for selection. Within the pZFN1 and pZFN2 plasmids (left), each ZFN monomer is placed under control of the inducible arabinose promoter, which allows fine-tuning of the expression level based on the concentration of arabinose in the culture medium. Each expression plasmid contains a different antibiotic resistance marker and also compatible low-copy replication origins. Within the pTox plasmid (at right) the highly lethal topoisomerase inhibitor ccdB is expressed under control of the T7 promoter and a compatible origin of replication. pTox also contains the ZFN dimer target, cleavage of which leads to plasmid clearance. The bacterial cells used in this study express T7 RNA polymerase under control of the lac promoter. The cells also express the lac inhibitor and T7 lysozyme. b Linker library design. The amino acid sequence of the host ZFN used for selection is shown in single letter code, with the FokI cleavage domain and ZFP regions indicated. The ZFP region is shown as an alignment of the four fingers with recognition helices highlighted in gray. The location and composition of the randomized linker is shown in red (N = mixture of all bases, S = mixture of G and C). The randomized linker library is inserted between the carboxy-terminal residue of the FokI nuclease domain and the first conserved residue in the zinc-finger domain. The library length varies from four to twenty-two residues. c Sketch of cleavage target used for selections. The bound ZFN dimer is shown consisting of the fixed right-hand ZFN (CCR5-L) and the left-hand ZFN bearing the linker library (CCR5-R) with randomized linker highlighted in red