Registry/Measurement kit/Protocols/Plate reader experiment: Difference between revisions
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'''This needs to be updated to reflect the measurement kit constructs''' | '''This needs to be updated to reflect the measurement kit constructs''' | ||
*be sure to include a negative control of TOP10 without plasmid. | *be sure to include a negative control of TOP10 without plasmid. | ||
#Three 5 ml cultures of supplemented M9 | #Three 5 ml cultures of supplemented M9 medium (w/glycerol) and antibiotic (kanamycin, 20 µg/ml) were inoculated with single colonies (~2mm ø) from a freshly streaked plate of MG1655 containing BBa_T9002. One 5 ml culture was inoculated with a single colony from a freshly streaked plate of MG1655 containing a BBa_T9002 mutant lacking a GFP expression device (T9002m) described in the stability section. | ||
#* | #* | ||
#Cultures were grown in 17 mm test tubes for 15 hrs at 37°C with shaking at 70 rpm. | #Cultures were grown in 17 mm test tubes for 15 hrs at 37°C with shaking at 70 rpm. |
Revision as of 15:11, 8 November 2007
This needs to be updated to reflect the measurement kit constructs
- be sure to include a negative control of TOP10 without plasmid.
- Three 5 ml cultures of supplemented M9 medium (w/glycerol) and antibiotic (kanamycin, 20 µg/ml) were inoculated with single colonies (~2mm ø) from a freshly streaked plate of MG1655 containing BBa_T9002. One 5 ml culture was inoculated with a single colony from a freshly streaked plate of MG1655 containing a BBa_T9002 mutant lacking a GFP expression device (T9002m) described in the stability section.
- Cultures were grown in 17 mm test tubes for 15 hrs at 37°C with shaking at 70 rpm.
- Cultures were diluted 1:1000 into 5.5 ml of pre-warmed fresh medium and grown to an OD600 of 0.15 under the same conditions as before. This growth took on average 4.5 hrs.
- Twenty-four 200 µl aliquots of each of the cultures were transferred into a flat-bottomed 96 well plate (Cellstar Uclear bottom, Greiner).
- 2ml of the stock concentrations of the cognate AHL, 3-oxohexanoyl-homoserine lactone (3OC6HSL), was added to each well to yield 8 different final concentrations (0, 1E-10, 1E-9, 1E-8, 1E-7, 1E-6, 1E-5 and 1E-4 M). Three replicate wells were measured for each concentration of 3OC6HSL. Three wells were each filled with 200 ml of medium to measure the absorbance background. Three further wells were each filled with 200 ml of the BBa_T9002 mutant culture to measure fluorescent background.
- 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. Approximately 6 min elapsed between beginning addition of 3OC6HSL to the wells and the first plate reader measurement. 3OC6HSL was added in order of increasing concentration to minimize GFP synthesis during plate loading. Cells appear to grow exponentially for the duration of the plate reader measurement protocol (see Supplementary Fig. 1 for representative growth curves).
- We repeated steps 1 through 6 on three separate days to obtain data for nine colonies from a single plate.
- Data processing was used to calculate the GFP synthesis rate for each well and is described in the data analysis section (below). The data for each colony tested was averaged across the three replicates wells. The mean for all colonies was then averaged to obtain a population mean. The time-dependent input-output surface is shown in Supplementary Figure 2. Following an initial transient response, device output reached an approximate steady state.
- The snapshot transfer function in Figure 1 is the 60 min time-slice from the surface shown in Supplementary Figure 1 (highlighted as a heavy black line). Error bars in Figure 1 represent the 95% confidence interval in the population for the nine independent samples. At this time point, the output of the device ranges from a minimum of 0 to a maximum of 521 molecules GFP cell-1 s-1. Characteristics of the snapshot transfer curve were defined relative to that maximum output.
- The Input Swing is defined as the minimum range of measured inputs for which device output swings from 5% to 95% of its maximum value.
- The Output Swing is defined as the range of population mean outputs over the input swing.
- The Switch Point is defined as the input level at which device output is 50% of the maximum output. This characteristic is calculated by linear interpolation between the measured data points. The uncertainty in the switch point was calculated by propagating the uncertainty in the measurement of the maximum output level through the switch point calculation.