Talk:Cconboy:Promoter Characterization/Methods

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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.
    • The construct is based on EYFP (E0030), which has a LVA tag. Is there any specific reason for that ? Why not choosing a more stable EYFP protein to limit fluctuations of the measurements once at steady state (rapid degradation) ?
      • E0030 does not have an LVA tag.[1]. The protein half-life of E0030 is on the order of days.
    • How many constructs were screened with this protocol ? Which ones?
      • N=16. The list of screened constructs is here.
  • 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.
    • Have you recorded the overnight OD600 ?
  • 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.
    • Have you also recorded the OD600 every 45 min?
    • From the measurement, after o/n and dilution, did you get the feeling that some of the promoters were shut-down because of the stationary phase ?
    • Is it with this protocol that you got the feeling that you have never been able to reach the steady state of EYFP expression (discussion section)?
  • 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 sample set included the 16 promoters available in the Registry as of 07-01-04.
    • Working at OD600=1.0 seems quite close to the stationary phase, as opposed to make sure that you are in exponential growth. It seems also quite different from the characterization of F2620 (OD600<0.4, with a GFP reporter). Is there any particular reason for that ?
  • 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.
    • Do you feel that you are at steady state in terms of EYFP expression ? Maybe you don't need to care to much about it.
  • 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.
  • Samples were prepared as above from 2mL of culture.
    • Have you done some experiments using a chemostat ? If yes, at which OD600 ?

Plate Reader Measurement

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