Negative selection

The negative selections were performed to eliminate the cohesin and the dockerin mutants that could interact with Doc_wt and Coh_wt, respectively. For negative selection with the cohesin library, WNPPI8 cells containing pBT-Coh_lib and pTRG-Doc_wt were cultivated in 5 mL LB medium overnight. The overnight culture (1 mL) was collected using a tabletop centrifuge at 2000 g for 10 minute at room temperature. After removal of the LB medium, cells were washed once using 1 mL of M9 complete medium (1 × M9 salts, 1 mM MgSO₄, 0.1 mM CaCl₂, 10 mg/L thiamine, 0.01 mM ZnSO₄, 0.2 mM uracil, 1 g/L histidine, 0.01% yeast extract, 0.4% D-glucose), then re-suspended in 1 mL of the same medium. The cells were incubated at 37 °C for 2 hours with shaking (225 rpm), which allowed cells to adapt to the growth in minimal medium before plating. Around 5 × 10⁶ cells were plated on M9 complete medium plates containing 2.5 mM 5-FOA, 0.05 mM IPTG, and appropriate concentrations of chloramphenicol and tetracycline. After 36 h of incubation at 37 °C, cells were collected from the plate into 1 mL M9 complete medium. Plasmid DNA was extracted from the collected cells. Mixture of the pBT-Coh_lib and pTRG-Doc_wt plasmids was first treated by restriction enzyme to linearize the pTRG-Doc_wt plasmid. The pBT-Coh_lib plasmids were then isolated by DNA gel electrophoresis purification. Negative selection with the dockerin library was conducted using the same procedure.