Registry/Measurement kit/Protocols/Plate reader experiment

This needs to be updated to reflect the measurement kit constructs

1. 1.For each test construct, a 5 ml culture of supplemented M9 medium (w/glycerol, see Endy Lab protocol) and antibiotic (kanamycin, 20 µg/ml) was inoculated with a single colony (~2mm ø) from a freshly streaked plate of MG1655 containing the construct. For each series, one 5 ml culture was inoculated with a single colony from a freshly streaked plate of TOP10 cells containing no vector. M9 media-supplemented without kanamycin was used for the empty TOP10 cells.
2. Cultures were grown in 17 mm test tubes for 18 hrs at 37°C with shaking at 70 rpm.
3. Cultures were diluted 1:1000 into 5 ml of pre-warmed fresh medium and grown for approximately 4.5 hours under the same conditions as before. No OD600 measurements were taken at this point.
4. For each culture a 200 µl aliquot was transferred into a flat-bottomed 96 well plate (Cellstar Uclear bottom, Greiner). An additional control of blank media was also added in the same aliquot. Three replicates of each culture/control were measured.
5. The plate was incubated in a Wallac Victor3 multi-well fluorimeter (Perkin Elmer) at 37°C and assayed with an automatically repeating protocol of absorbance measurements (600 nm absorbance filter, 0.1 second counting time through 5 mm of fluid), fluorescence measurements (488 nm excitation filter, 525 nm emission filter, 0.5 seconds, CW lamp energy 12902 units), and shaking (1 mm, linear, normal speed, 5 seconds). Time between repeated measurements was 2 min and 21 s.
6. We repeated steps 1 through 6 on over the course of several weeks.
7. Data processing was used to calculate the GFP/RFP synthesis rates for each well. The data for each colony tested was averaged across the three replicates wells.