InterLab

Calibration

This year, the goal of the iGEM InterLab Study is to answer the question: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?

Before starting the experiments, our team calibrated the necessary equipment we would need for the InterLab study.

The cells in table 1 to the right represent the plate wells, and all values are measured in μM.

Table 1. Calibration of the plate reader using serial dilutions of fluorescein.

Table 2 to the shows our microsphere calibration measured at 630 nm. Table 3 shows our absorbance calibration measured at 630nm. The cells in the table represent the plate wells.

Table 2. Microsphere calibration to determine a beads to cells conversion.

Table 3. Absorbance calibration measured at 630nm. All values are measured in abs.

Figure 1 and Figure 2 are calibration curves that show the means at each concentration of fluorescein. Figure 1 is shown with a standard scale, while Figure 2 is shown with a log scale.

Figure 1. Standard scale comparison of the arithmetic mean of the concentration of fluorescein to the fluorescence.

Figure 2. Log scale comparison of the arithmetic mean of the concentration of fluorescein to the fluorescence.

Figure 3 and Figure 4 are calibration curves that show the arithmetic mean of the number of particles compared to the absorbance at OD600. Figure 3 is shown with a standard scale, while Figure 4 is shown with a log scale.

Figure 3. Standard scale comparison of the arithmetic mean of the particle count/100 uL to the absorbance at OD600.

Figure 4. Log scale comparison of the arithmetic mean of the particle count/100 uL to the absorbance at OD600.

Plate Reader Data

Absorbance

Table 4. Our raw OD600 measurements of our samples at hour 0.

Table 5. Our raw OD600 measurements of our samples at hour 6.

Fluorescence

Table 6. Our raw fluorescence measurements of our samples at hour 0.

Table 7. Our raw fluorescence measurements of our samples at hour 6.

Figure 5 shows the normalized fluorescence data for each device, which was calculated by taking the fluorescence measurements divided by the OD600 measurements after 6 hours.. In the figure, the data for each sample is grouped together by device. Device 1 and Device 5 both have large variations across their data ranges, which could mean that these devices are less accurate than the others that have much more compact data. A different device, such as Device 2 or Device 6, could prove more accurate in finding a conversion between absorbance and fluorescence for different cell populations according to our data.

Figure 5. The normalized fluorescence data for each device.

CFU Data

Table 8 represents our data from the CFU portion of InterLab. Samples of cells with an OD600 of ~0.1 were diluted and grown on LB + chloramphenicol plates overnight. The next morning, the colonies were counted and used to predict the number of colony forming units in 1 mL of media.

Table 8. Our counted colonies per plate as well as calculated CFUs/mL. TNTC stands for Too Numerous to Count and indicates >300 colonies on the plate.

Figure 6. CFUs/mL vs. OD600 for our positive control cultures (BBa_I20270) and negative control cultures (BBa_R0040) samples.

Figure 6 to the left plots the absorbance of each sample in the plate reader against its calculated CFU/mL value after plating the different dilutions. We noticed a general positive correlation between the variables, which makes sense considering a greater number of cells should have a higher absorbance value. We noticed more variability in the positive cultures than the negative cultures. The 3 positive cultures in the bottom left portion of the data are all samples from the first positive culture and the 3 in the top right portion of the data are the three samples from the second positive culture.

Figure 7. The calculated conversion factors for determining CFUs/mL from an absorbance reading for each of our 12 samples.

The box and whiskers plot to the left shows the variation in our calculated conversion factor to convert from an OD600 value to a CFU/mL value. It was determined by taking the calculated CFU values for each of the 12 samples and dividing by that sample's OD600 value as determined by the plate reader. The median values between both the positive control and negative control samples are close, suggesting little variability between a positive and negative sample when determining CFU calculations from an OD600 measurement.

Team:RHIT/InterLab

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Design

Experiments

Notebook

Results