We used the Tecan InfiniteM200 plate reader to measure optical density and GFP fluorescence of small liquid cultures of cells containing our wild-type and mutant promoter screening constructs. We first cultured our samples in a 96-well plate or in 5 mL Falcon round-bottom polypropylene tubes 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 it with 5 uL of starter culture for screening runs and 15 uL for promoter characterization runs. We were sure to include both positive (Wildtype promoter) and negative (LB, DH5alpha) controls in each plate. All measurements for characterization runs 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. We can fit two data runs in our plate reader every 24 hours - 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 average fluorescence values for each mutant and culture condition by cell density (OD600), then normalized by dividing by the average fluorescence of our wild-type controls.

We corrected mutant and wild-type OD values for the absorptive properties of our media by subtracting the average OD of our LB control wells from sample ODs. We also corrected for non-GFP related cell fluorescence by subtracting the fluorescence readings of our DH5-α control from sample values.

Error bars were generated by adding and subtracting one standard deviation from the mean value measured at each point.

LacI Promoter

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