Figure 3. Construction of F′-based reporter episomes required for B2H selection strains (figure and legend adapted from Thibodeau-Beganny & Joung, 2007⁷⁵).

(a) Identical lacI^q and lacZ sequences present on both the F′ episome from strain CSH100 and the pKJ1712-derived reporter plasmid permit transfer of a cassette harboring a kanamycin resistance gene (Kan^R; orange box), target DNA site (black box), B2H promoter (arrow), and the co-cistronic HIS3 and aadA selectable markers (green boxes) from the plasmid to the F′ by a double recombination event (depicted by dashed lines). Note that the desired double-recombinant F′ would not harbor the counter-selectable sacB marker gene (red box) present on the reporter plasmid.

(b) Schematic depicting the various types of cells described in the bacterial mating of step 24. The left side of the figure depicts CSH100 cells in which a double, single, or no recombination event has occurred between the F′ and the reporter plasmid. The right side of the figure depicts KJ1C cells that have received a double, single, or non-recombinant F′ from the CSH100 cells and indicates whether or not these various cells will grow on LB/TKS plates containing tetracycline, kanamycin, and sucrose.