Part:BBa_I744102:Experience

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UNIQ6019100cc5232a55-partinfo-00000000-QINU UNIQ6019100cc5232a55-partinfo-00000001-QINU The following review was made by Michael Pettigrew:

The graph above is one of three measurements made. The others followed the same trend. The red points are data points, while the black line is a Hill Activator Equation fit to the experimental data.

Protocol:

1. Grow an overnight culture of BBa_I744102 (2mL LB and 100ug/mL ampicillin), as well as an overnight culture of BBa_J23119 to use as a negative control with no fluorescence. Both of these plasmids were in strain DH5a.

2. Prepare 10 tubes of 2mL LB + amp. Inoculate 9 with 20uL of I744102 overnight culture (1/100 dilution). Inoculate 1 tube with 20uL of J23119. Incubate at 37 degrees, 225 rpm until the OD600 of the cells is ~0.2.

3. Induce the 9 tubes of I744102 with the following concentrations of anhydrotetracycline (all in ng/mL): 0, 0.5, 1, 3, 5, 10, 25, 50, 150.

4. Incubate at 37 degrees, 225 rpm for 17.5 hours. If the cells were incubated for much less time than this, fluorescence wasn’t noticeable on our fluorometer. If they were incubated for much longer than this, EYFP accumulated in stationary phase and fluorescence became ATc-independent.

5. Measure fluorescence and OD600 in ocean optics spectrometer and fluorometer. The wavelength of the laser used in the fluorometer is 535.25nm, and the wavelength measured was 555.09nm. Perform 3 measurements at each ATc concentration (600uL per cuvette).