This year's interlab study focused on the measuring fluorescence of specific Igem devices. The first step was calibrating the measurements by creating a reference point of optical density. Four samples of LUDOX-S30 and four samples of deionized water. The protocol called for the optical density of the samples to be measured at 600 nm, but due to the type of plate reader used, the optical density was measured at 590 nm. The plate reader was unable to measure samples accurately at 600nm.
Table 1:Optical Density 590nm Reference Point
The optical density reference point was measured and shown to be around 0.0395 at 590nm for the Ludox and 0.0375 for the water. The correction factor of 7.375 created from the measurements was used in later calculations.
Next a standard curve was created for the fluorescence of samples using a serial dilution of FITC in 1x PBS. This was done four times and the average values for each concentration were used to create the graph shown beneath the table.
Table 2:FITC Standard Curve Measurements Figure 1:Standard Curve of Average FITC Serial Dilutions
The standard curve of the FITC serial dilutions was created and created a trend line that was close to being linear, having fluorescence increase proportionally to the increase in concentration. This standard curve was used to analyze the measurements of fluorescence of the Interlab test devices.
After the liquid cultures of the test devices was grown, a sample from each was taken and the optical density was measured in a spectrophotometer at 600nm. This measurment was then used to back dilute the samples into a 10 mL volume of LB media and chloramphenicol
Table 3:Normalization of Device Optical Density
The volumes shown in the table were used to create the 10mL stocks that were then left to incubate at 37 Celcius. Every hour, one mL of stock was taken and chilled on ice that were measured in the plate reader at the end
The absorbency of the samples collected of the positive control, negative control, and test devices 1 through 3 from each hour collected over a six hour period were measured at 590nm
Table 4:Absorbency and Correction of InterLab Plate Table 5:Corrected Absorbency of Interlab Devices
Shown in Table 4 and Table 5, the devices showed a trend of gradual increase in absorbency overtime, with the highest absorbencies measured at the six hour time period. The corrected values were then plotted to better portray the trend of the data collected, in Figure 2.
Figure 2:InterLab Absorbency Graph
The graph showed that the negative control had the highest absorbency at the six hour mark. Test device 1 for both of its replicates had the lowest absorbency for all the time periods.
The fluorescence of each of the devices was also measured with the normalized plate reader, measuring emission at 485nm and absorption at 535nm. The results from the plate reader were first recorded in the top table below. The data were then adjusted to subtract out the pre-recorded absorbency of the blank (the media the cells were grown in).
InterLab Fluorescence Table
It was observed in the tables that device two had the highest levels of fluorescence, followed by the positive control, then the first device. The negative control samples showed the lowest levels of fluorescence out of all of the samples collected. The data collected was then organized in a graph to better depict the possible differences in absorbances between the samples. The graph displayed the data as fluorescence in absorbency units over time the sample was incubated for in hours
InterLab Fluorescence Graph
The graph created in the figure above matched the observations made from its related graph. It also showed that each sample had defined trends of fluorescence and all but the negative control increased in fluorescence as incubation time increased.
InterLab Fluorescence/Abs590 Tables
The results of the calculations made above were then used to plot the relation between fluorescence verse optical density and show just how much the data varied for each sample and for each incubation time
InterLab Fluorescence/Abs590 Graph
The figure above showed that device 1 had the highest values for the relation between fluorescence and optical density. In addition, time zero for the first device had the highest variation. Times 4 hours and 6 hours had the lowest levels of variation for most sample devices used in the interlab.