Cconboy:Promoter Characterization/Methods

Promoter activity time course

• Cultures of LB with 50ug/mL Amp were inoculated from fresh plates of MC4100 containing one of a select set of promoter characterization constructs.
• Cultures were grown overnight to saturation at 37∞C, then diluted 1:500 into 50mL fresh LB/amp at the beginning of the time course.
• Samples were collected for flow cytometry from the saturated overnights (2mL), at the time of dilution (10mL), and every 45 min thereafter for six hours. (10mL at t=0.75 hr; 5mL at t=1.25 and 2 hr; 2mL thereafter.) An additional sample was drawn at the tenth hour.
• All samples were spun down, and collected cells were resuspended in 0.5 or 1mL of PBS (according to cell yield) and stored on ice until the tenth hour. A control experiment showed that reserving samples on ice for up to 12 hours did not affect the level of YFP detected by flow cytometry significantly. Variation of +/- 5% was observed. (Data not shown.)
• YFP fluorescence of samples was measured by flow cytometry.

Promoter activity “snapshots”

Based on the curves generated by the promoter activity time course experiments, OD600=1.0 was selected as the condition for making a single measurement of YFP output for the complete set of promoter characterization constructs. This 32-sample set includes all promoters available in the Registry as of 07-01-04.

Overnight cultures were prepared as above, diluted 1:100 into 5mL fresh LB/amp, and grown for 2.5 hours at 37∞C before sampling. The average OD600 at the time of sampling was 1.12, with a standard deviation of 0.16, excluding one sample (I6058) which exhibited a severe growth delay. I6058 was sampled at 4.5 hrs post-dilution when it reached OD600=0.91. (See discussion.) Samples were prepared as above from 2mL of culture.