To collect our data, we first cultured our samples in a 96-well plate for six hours at 37 C, or 12 hours at room temperature. This allowed us to ensure that all of our samples were in exponential phase at the start of the experiment, and that the growth curves would be synchronized across wells.

We then filled a second 96-well plate with media of various conditions, and inoculated with 15 uL of culture from the culture plate. We were sure to include both positive (Wildtype promoter) and negative (LB, DH5alpha) controls. All measurements were performed in triplicate.

For our R0010 mutants, we had to collect data with two varying parameters: IPTG concentration and Arabinose concentration. Each plate has enough wells to test a single construct under 28 different conditions in triplicate with controls, allowing us to simultaneously test four different IPTG and seven different Arabinose levels. Working efficiently, we are able to fit two data runs in our fluorescence reader per day - that's two complete mutant characterizations in a single day!

For other mutants, the room on the plate allows us to test four different constructs under seven different conditions, allowing us to characterize large libraries in a very short time.

To obtain our relative fluorescence values, we divided the raw fluorescence values by cell density, then normalized against the wildtype control.

LacI Promoter

Click here to view the fluorescence characterization results for wild-type and mutant LacI-repressible promoters!