Figure 4.

Orthogonal substrate usage observed between mutants 53 and 54. Mutant 53 produces more light with disubstituted luciferins than singly modified analogs. Bacteria expressing mutant 53, mutant 54, or Fluc were lysed and incubated with 4’-Br-7’-MeLuc or a singly modified luciferin at saturating doses (100–250 μM). The light output for each enzyme-substrate reaction was measured. Comparative emission values are plotted as the ratio of disubstituted luciferin activity over the activity of each substrate shown, on a log scale. Error bars represent the standard error of the mean for n ≥ 3 experiments. For each replicate, a single lysate was split among three wells and treated with the various analogs.