Contributors: Esther, Chloe, Alexander
WT CxnA beta-glucosidase selection assay
Serial dilution (1:10, 1:100, 1:1000) of transformed CxnA culture in PBS. Plated in triplicate across conditions (undiluted, 1:100, 1:1000 — 1:10 omitted due to plate limits). Incubated at 37 °C from 16:00 on 24th June; to check at 48 hours (carbon-source-only selection requires extended incubation).
Goal: confirm WT CxnA has no beta-glucosidase activity before beginning directed evolution.
CMC plates — endoglucanase activity control
LB agar + chloramphenicol (40 µg/mL) + IPTG (0.38 mM) + CMC (0.2% w/v). Not autoclaved before additions (added CMC, microwaved to dissolve, then IPTG/chloramphenicol). 12 plates poured, stored in fridge, not yet used.
Protocol: Streak transformants; incubate overnight 37 °C; flood with 0.1% Congo Red 15 min; wash with 1 M NaCl 15 min; examine for zones of clearance (endoglucanase activity degrades CMC and clears the red stain).
MUC plates — exoglucanase activity control
LB agar + chloramphenicol (40 µg/mL) + IPTG (0.38 mM), autoclaved before additions. 12 plates poured; 100 µl of 10 mM MUC spread over plate surface (50.05 mg MUC in 1 ml DMSO); 10 of 12 plates covered. Stored in fridge, not yet used.
Protocol: Streak colonies; incubate overnight 37 °C; expose to 364 nm UV lamp (MUC substrate fluoresces upon cleavage by exoglucanase activity).
Additional plating
CxnA plated on two spare chloramphenicol + IPTG plates (1 undiluted, 1 at 1:100 dilution) as a supplementary control.