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Engineering Cys2His2 Zinc Finger Domains 325Fig. 4. Homologous recombination between reporter plasmids and the CSH100 strainF′ mediated by regions of sequence identity. Schematic of a reporter plasmid showing itsstructure and its regions of sequence identity with the F′ from E. coli strain CSH100. Adouble homologous recombination event between the two regions of sequence identity(from the lacI q and lacZ genes) leads to insertion of a fragment consisting of the Kan Rgene, the target DNA-binding site, and the HIS3/aadA reporter into the F′. Note that adouble homologous recombination event does not transfer the counter-selectable sacBgene from the reporter plasmid to the F′.2. Vortex to resuspend the CSH100 transformants (see Note 6) and transferapprox 200 µL of this cell resuspension to a fresh 25-mm tube with 5 mL of LB(see Note 7).3. Transfer approx 200 µL of an overnight culture of KJ1C cells (inoculated the nightbefore, grown in LB containing 12.5 µg/mL of tetracycline) to a sterile 25-mmtube containing 10 mL of LB.4. Prepare a control 25-mm tube containing 10 mL of LB.5. Incubate all tubes from steps 2 to 4 for 2 h at 37°C without agitation, therebyallowing cells to grow to log phase and for CSH100 cells to form F pili.6. To perform matings, mix together the following combinations of the cultures fromsteps 2 to 4 in sterile 18-mm glass tubes. Use 1 mL of each liquid culture (i.e., eachmating will consist of a total of 2 mL).