Difference between revisions of "Team:RHIT/InterLab"
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<h1>Calibration</h1> | <h1>Calibration</h1> | ||
| − | <p> Before starting the experiments, our team calibrated the necessary equipment we would need for the InterLab study. </p> | + | <p> 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? <br><br> |
| + | Before starting the experiments, our team calibrated the necessary equipment we would need for the InterLab study. </p> | ||
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| + | <p> 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.</p> | ||
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<img src = "https://static.igem.org/mediawiki/2018/2/28/T--RHIT--MicrosphereCalibration.jpg"> | <img src = "https://static.igem.org/mediawiki/2018/2/28/T--RHIT--MicrosphereCalibration.jpg"> | ||
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| + | <img src = "https://static.igem.org/mediawiki/2018/3/39/T--RHIT--AbsorbanceCalibration.jpg" style="width:70%"></center> | ||
| + | <center>Table 3. Absorbance calibration measured at 630nm. All values are measured in abs. </center> | ||
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<p> 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. </p> | <p> 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. </p> | ||
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<p> 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. </p> | <p> 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. </p> | ||
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| − | Figure 5 shows the normalized fluorescence data for each device. In the figure the data for each | + | <p>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.</p> |
<center> | <center> | ||
<img src = "https://static.igem.org/mediawiki/2018/e/ea/T--RHIT--InterLabGraph.JPG"> | <img src = "https://static.igem.org/mediawiki/2018/e/ea/T--RHIT--InterLabGraph.JPG"> | ||
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<h1> CFU Data </h1> | <h1> CFU Data </h1> | ||
<p> 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. </p> | <p> 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. </p> | ||
<img src = "https://static.igem.org/mediawiki/2018/0/0a/T--RHIT--CFUInterLab.jpg"> | <img src = "https://static.igem.org/mediawiki/2018/0/0a/T--RHIT--CFUInterLab.jpg"> | ||
| − | <center> Table 8. Our counted colonies per plate as well as calculated CFUs/mL.</center | + | <center> 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. </center> |
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<img src = "https://static.igem.org/mediawiki/2018/b/b1/T--RHIT--CFUgraph2.jpg"> | <img src = "https://static.igem.org/mediawiki/2018/b/b1/T--RHIT--CFUgraph2.jpg"> | ||
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<p> 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. </p> | <p> 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. </p> | ||
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<img src = "https://static.igem.org/mediawiki/2018/2/2a/T--RHIT--CFUConversion2.jpg"> | <img src = "https://static.igem.org/mediawiki/2018/2/2a/T--RHIT--CFUConversion2.jpg"> | ||
| − | <center> Figure 7. The calculated conversion factors for determining CFUs/mL from an absorbance reading. </center> | + | <center> Figure 7. The calculated conversion factors for determining CFUs/mL from an absorbance reading for each of our 12 samples. </center> |
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<p> 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. </p> | <p> 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. </p> | ||
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Latest revision as of 14:12, 8 August 2018
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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 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.
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
Fluorescence
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.
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.
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.