Bl21 de3 cells

Spot titer tests were performed to determine the level of β-lactam antibiotic resistance of cells expressing the β-lactamase tripartite fusions. A single colony from fresh E. coli BL21 (DE3) cells (Stratagene) (transformed with the appropriate plasmid) was used to inoculate 100 mL sterile LB containing 10 μg/mL tetracycline (Formedium). Cultures were incubated overnight at 37 °C with shaking (200 rpm). 1 mL of overnight culture was used to inoculate 100 mL sterile LB containing 10 μg/mL tetracycline and grown at 37 °C (shaking at 200 rpm) until an OD₆₀₀ of 0.6 was reached. Expression of the β-lactamase fusion construct was induced by the addition of filter-sterilized arabinose (Sigma) to a final concentration of 0.02 % (w/v). Cultures were incubated for a further 1 h, when the OD₆₀₀ of the cells was adjusted to 1.0 using sterile LB, and the cultures were then serially diluted in 10-fold increments into sterile 170 mM NaCl solution. 3 μL of each dilution was then spotted onto LB agar plates, supplemented with 10 μg/mL tetracycline, 0.02 % (w/v) arabinose, and increasing concentrations of ampicillin (Formedium) (0–140 μg/mL). The plates were incubated at 37 °C for 18 h and the maximal cell dilution allowing cell growth (MCD_GROWTH) was determined for each ampicillin concentration by visual inspection.