Introduction
The iGEM InterLab Measurement Study is designed around the idea of developing a way to standardize measurement of fluorescence among several synthetic biology labs across the world. By completing the 2018 iGEM InterLab Study, Purdue iGEM is a part of an entire international force that has combined to continue that goal. This year’s interlab study involves reducing variability of fluorescent measurements by using colony-forming units (CFUs) rather than using optical density (OD).
Characteristics of the FlexStation 3
The plate reader used for Purdue iGEM’s InterLab Study was the Molecular Devices FlexStation 3. This modeluses top optics to read both absorbance and fluorescence. The device includes a pathlength correction ability, but it was disabled for conducting the InterLab Study. The device also has the ability to adjust the temperature before the plate is read, but all plate readings were conducted at room temperature. When measuring fluorescence, a filter with a 530nm/ 30nm bandpass, 25nm bandpass width, 485nm excitation, and 525nm emission was used. Black 96 well plates with transparent, flat bottoms were used for each of the measurements.
Calibrations
Cell Measurement Protocol
The cell measurement protocol was followed as defined in the iGEM 2018 InterLab Study Protocol with the exception of the Colony Forming Units per 0.1 OD600 E. Coli Cultures Step 2: Dilution Series protocol. Instead of using 2mL microcentrifuge tubes, only 1.5mL tubes were used. Therefore, all volumes used in the serial dilution were halved. However, 100uL of dilutions 3, 4, and 5 were still used for plating as described in the protocol.
Results
OD 600 Reference Point
Particle Standard Curve
Fluorescein Standard Curve
Raw Plate Reader Measurements
Fluorescence Raw Readings: | Abs600 Raw Readings: | ||||||||||||||||||||
Hour 0: | Neg. Contr | Pos. Contr | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) | Hour 0: | Neg. Contr | Pos. Contr | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) | ||
Colony 1, Replicate | 3.8675 | 78.0275 | 175.3375 | 96.3575 | -6.7025 | 219.2175 | 121.7075 | 40.9775 | 0.3275 | Colony 1, Replicate | 0.01725 | 0.02625 | 0.01625 | 0.01625 | 0.01725 | 0.01425 | 0.01125 | 0.01525 | 0.00025 | ||
Colony 1, Replicate | 6.8075 | 69.7375 | 156.0075 | 135.6975 | -6.5625 | 219.7075 | 111.1175 | 40.6875 | -2.6225 | Colony 1, Replicate | 0.01525 | 0.01425 | 0.01325 | 0.01425 | 0.01925 | 0.01525 | 0.01025 | 0.01625 | 0.00025 | ||
Colony 1, Replicate | 4.4175 | 72.8675 | 144.2075 | 84.0775 | -12.0925 | 226.2175 | 96.2175 | 37.1075 | 1.2675 | Colony 1, Replicate | 0.01425 | 0.01325 | 0.01125 | 0.01725 | 0.01425 | 0.01625 | 0.00925 | 0.01325 | 0.00225 | ||
Colony 1, Replicate | 5.3375 | 77.6475 | 190.9275 | 88.1575 | -6.1825 | 253.0975 | 95.0875 | 41.6775 | 1.9975 | Colony 1, Replicate | 0.01625 | 0.01425 | 0.01425 | 0.01525 | 0.01525 | 0.04225 | 0.01025 | 0.01425 | -0.00075 | ||
Colony 2, Replicate | 5.7075 | 57.0875 | 166.5475 | 120.5275 | -0.8325 | 209.8475 | 124.3075 | 23.2075 | -0.7125 | Colony 2, Replicate | 0.01425 | 0.01425 | 0.01925 | 0.01525 | 0.01725 | 0.01325 | 0.01225 | 0.00825 | 0.00025 | ||
Colony 2, Replicate | 6.4375 | 64.7975 | 123.7975 | 115.4075 | -2.1425 | 158.4275 | 107.7175 | 23.7275 | -2.2425 | Colony 2, Replicate | 0.02525 | 0.02025 | 0.01125 | 0.01525 | 0.01425 | 0.06025 | 0.01025 | 0.01525 | -0.00075 | ||
Colony 2, Replicate | 0.0075 | 54.7275 | 167.3475 | 127.5475 | -3.5425 | 173.3975 | 102.6375 | 19.4575 | 2.2475 | Colony 2, Replicate | 0.01525 | 0.01925 | 0.03825 | 0.01425 | 0.01425 | 0.01225 | 0.01025 | 0.01325 | -0.00075 | ||
Colony 2, Replicate | 9.7775 | 64.9275 | 125.1075 | 139.2775 | -12.9025 | 175.6275 | 129.8875 | 20.2475 | -0.2625 | Colony 2, Replicate | 0.01225 | 0.01325 | 0.00825 | 0.01625 | 0.01525 | 0.01025 | 0.01225 | 0.01425 | -0.00075 | ||
Hour 6: | Neg. Contr | Pos. Contr | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) | Hour 6: | Neg. Contr | Pos. Contr | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) | ||
Colony 1, Replicate | 45.4775 | 1983.898 | 363.8275 | 2256.998 | 53.5275 | 686.8375 | 194.1975 | 544.8175 | 9.6475 | Colony 1, Replicate | 0.43375 | 0.31375 | 0.01975 | 0.43775 | 0.40375 | 0.02575 | 0.02375 | 0.31775 | -0.00025 | ||
Colony 1, Replicate | 27.2675 | 1958.998 | 377.7875 | 2168.898 | 44.9875 | 745.2175 | 191.4975 | 555.6675 | -11.3325 | Colony 1, Replicate | 0.39775 | 0.25675 | 0.01675 | 0.44275 | 0.40175 | 0.02775 | 0.01175 | 0.30675 | 0.00275 | ||
Colony 1, Replicate | 38.7975 | 2054.698 | 328.0475 | 1760.998 | 25.6075 | 760.1675 | 181.9875 | 543.6175 | -7.6925 | Colony 1, Replicate | 0.42175 | 0.27375 | 0.01575 | 0.32275 | 0.31675 | 0.02975 | 0.01575 | 0.31175 | 0.00375 | ||
Colony 1, Replicate | 40.4975 | 2051.698 | 308.0075 | 2095.498 | 46.1175 | 743.3175 | 228.3375 | 667.3075 | -5.8425 | Colony 1, Replicate | 0.41375 | 0.25475 | 0.01475 | 0.38975 | 0.39675 | 0.02975 | 0.01675 | 0.35375 | 0.00975 | ||
Colony 2, Replicate | 46.5475 | 1235.198 | 240.2175 | 2156.898 | 65.5375 | 387.5275 | 362.6575 | 673.0775 | -2.2225 | Colony 2, Replicate | 0.43075 | 0.35175 | 0.00975 | 0.34775 | 0.45275 | 0.01975 | 0.02575 | 0.35775 | -0.00325 | ||
Colony 2, Replicate | 43.6975 | 1510.898 | 309.5175 | 2124.598 | 60.9975 | 407.4775 | 453.4975 | 853.0275 | 9.4875 | Colony 2, Replicate | 0.48375 | 0.32875 | 0.01275 | 0.44775 | 0.49475 | 0.01275 | 0.02975 | 0.47075 | -0.00325 | ||
Colony 2, Replicate | 38.0875 | 1617.498 | 302.7175 | 2604.498 | 59.4275 | 384.8775 | 406.2575 | 856.5075 | 5.8775 | Colony 2, Replicate | 0.47375 | 0.41975 | 0.01275 | 0.41275 | 0.47875 | 0.01275 | 0.02875 | 0.46675 | -0.00425 | ||
Colony 2, Replicate | 53.5775 | 1604.198 | 258.8175 | 2834.498 | 51.4375 | 354.0175 | 452.7775 | 907.7975 | 2.0775 | Colony 2, Replicate | 0.46875 | 0.42675 | 0.01175 | 0.45075 | 0.48275 | 0.01275 | 0.02775 | 0.43675 | -0.00525 |
Flourescense per OD
µM Fluorescein / OD | Net Fluorescein a.u. | Net Abs 600 | ||||||||||||||||||||||||
Hour 0: | Neg. Contr | Pos. Contr | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | Neg. Contr | Pos. Contr | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | Neg. Contr | Pos. Contr | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | ||
Colony 1, Replicate 1 | 0.017 | 0.242 | 0.884 | 0.485 | -0.033 | 1.264 | 0.892 | 0.219 | 3.54 | 77.70 | 175.01 | 96.03 | -7.03 | 218.89 | 121.38 | 40.65 | 0.017 | 0.026 | 0.016 | 0.016 | 0.017 | 0.014 | 0.011 | 0.015 | ||
Colony 1, Replicate 2 | 0.051 | 0.418 | 0.986 | 0.798 | -0.017 | 1.198 | 0.919 | 0.219 | 9.43 | 72.36 | 158.63 | 138.32 | -3.94 | 222.33 | 113.74 | 43.31 | 0.015 | 0.014 | 0.013 | 0.014 | 0.019 | 0.015 | 0.010 | 0.016 | ||
Colony 1, Replicate 3 | 0.021 | 0.526 | 1.284 | 0.446 | -0.090 | 1.299 | 1.096 | 0.263 | 3.15 | 71.60 | 142.94 | 82.81 | -13.36 | 224.95 | 94.95 | 35.84 | 0.012 | 0.011 | 0.009 | 0.015 | 0.012 | 0.014 | 0.007 | 0.011 | ||
Colony 1, Replicate 4 | 0.016 | 0.408 | 1.018 | 0.435 | -0.041 | 0.472 | 0.684 | 0.214 | 3.34 | 75.65 | 188.93 | 86.16 | -8.18 | 251.10 | 93.09 | 39.68 | 0.017 | 0.015 | 0.015 | 0.016 | 0.016 | 0.043 | 0.011 | 0.015 | ||
Colony 2, Replicate 1 | 0.037 | 0.334 | 0.711 | 0.653 | -0.001 | 1.309 | 0.842 | 0.242 | 6.42 | 57.80 | 167.26 | 121.24 | -0.12 | 210.56 | 125.02 | 23.92 | 0.014 | 0.014 | 0.019 | 0.015 | 0.017 | 0.013 | 0.012 | 0.008 | ||
Colony 2, Replicate 2 | 0.027 | 0.258 | 0.849 | 0.594 | 0.001 | 0.213 | 0.808 | 0.131 | 8.68 | 67.04 | 126.04 | 117.65 | 0.10 | 160.67 | 109.96 | 25.97 | 0.026 | 0.021 | 0.012 | 0.016 | 0.015 | 0.061 | 0.011 | 0.016 | ||
Colony 2, Replicate 3 | -0.011 | 0.212 | 0.342 | 0.675 | -0.031 | 1.064 | 0.738 | 0.099 | -2.24 | 52.48 | 165.10 | 125.30 | -5.79 | 171.15 | 100.39 | 17.21 | 0.016 | 0.020 | 0.039 | 0.015 | 0.015 | 0.013 | 0.011 | 0.014 | ||
Colony 2, Replicate 4 | 0.062 | 0.376 | 1.126 | 0.663 | -0.064 | 1.292 | 0.809 | 0.111 | 10.04 | 65.19 | 125.37 | 139.54 | -12.64 | 175.89 | 130.15 | 20.51 | 0.013 | 0.014 | 0.009 | 0.017 | 0.016 | 0.011 | 0.013 | 0.015 | ||
Hour 6: | Neg. Contr | Pos. Contr | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | Neg. Contr | Pos. Contr | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | Neg. Contr | Pos. Contr | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | ||
Colony 1, Replicate 1 | 0.007 | 0.508 | 1.431 | 0.415 | 0.009 | 2.105 | 0.621 | 0.136 | 35.83 | 1974.25 | 354.18 | 2247.35 | 43.88 | 677.19 | 184.55 | 535.17 | 0.434 | 0.314 | 0.020 | 0.438 | 0.404 | 0.026 | 0.024 | 0.318 | ||
Colony 1, Replicate 2 | 0.008 | 0.627 | 2.246 | 0.400 | 0.011 | 2.446 | 1.821 | 0.151 | 38.60 | 1970.33 | 389.12 | 2180.23 | 56.32 | 756.55 | 202.83 | 567.00 | 0.395 | 0.254 | 0.014 | 0.440 | 0.399 | 0.025 | 0.009 | 0.304 | ||
Colony 1, Replicate 3 | 0.009 | 0.617 | 2.261 | 0.448 | 0.009 | 2.387 | 1.277 | 0.145 | 46.49 | 2062.39 | 335.74 | 1768.69 | 33.30 | 767.86 | 189.68 | 551.31 | 0.418 | 0.270 | 0.012 | 0.319 | 0.313 | 0.026 | 0.012 | 0.308 | ||
Colony 1, Replicate 4 | 0.009 | 0.679 | 5.073 | 0.447 | 0.011 | 3.027 | 2.704 | 0.158 | 46.34 | 2057.54 | 313.85 | 2101.34 | 51.96 | 749.16 | 234.18 | 673.15 | 0.404 | 0.245 | 0.005 | 0.380 | 0.387 | 0.020 | 0.007 | 0.344 | ||
Colony 2, Replicate 1 | 0.009 | 0.282 | 1.507 | 0.497 | 0.012 | 1.369 | 1.017 | 0.151 | 48.77 | 1237.42 | 242.44 | 2159.12 | 67.76 | 389.75 | 364.88 | 675.30 | 0.434 | 0.355 | 0.013 | 0.351 | 0.456 | 0.023 | 0.029 | 0.361 | ||
Colony 2, Replicate 2 | 0.006 | 0.365 | 1.515 | 0.379 | 0.008 | 2.010 | 1.087 | 0.144 | 34.21 | 1501.41 | 300.03 | 2115.11 | 51.51 | 397.99 | 444.01 | 843.54 | 0.487 | 0.332 | 0.016 | 0.451 | 0.498 | 0.016 | 0.033 | 0.474 | ||
Colony 2, Replicate 3 | 0.005 | 0.307 | 1.411 | 0.504 | 0.009 | 1.802 | 0.981 | 0.146 | 32.21 | 1611.62 | 296.84 | 2598.62 | 53.55 | 379.00 | 400.38 | 850.63 | 0.478 | 0.424 | 0.017 | 0.417 | 0.483 | 0.017 | 0.033 | 0.471 | ||
Colony 2, Replicate 4 | 0.009 | 0.300 | 1.221 | 0.502 | 0.008 | 1.580 | 1.104 | 0.166 | 51.50 | 1602.12 | 256.74 | 2832.42 | 49.36 | 351.94 | 450.70 | 905.72 | 0.474 | 0.432 | 0.017 | 0.456 | 0.488 | 0.018 | 0.033 | 0.442 |
Flourescense per Particle
Hour 0: | Neg. Contr | Pos. Contr | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | Neg. Contr | Pos. Contr | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | Neg. Contr | Pos. Contr | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | ||
Colony 1, Replicate 1 | 1.63E+02 | 2.34E+03 | 8.57E+03 | 4.70E+03 | -3.24E+02 | 1.22E+04 | 8.64E+03 | 2.12E+03 | 3.54 | 77.70 | 175.01 | 96.03 | -7.03 | 218.89 | 121.38 | 40.65 | 0.017 | 0.026 | 0.016 | 0.016 | 0.017 | 0.014 | 0.011 | 0.015 | ||
Colony 1, Replicate 2 | 4.92E+02 | 4.05E+03 | 9.56E+03 | 7.74E+03 | -1.62E+02 | 1.16E+04 | 8.91E+03 | 2.12E+03 | 9.43 | 72.36 | 158.63 | 138.32 | -3.94 | 222.33 | 113.74 | 43.31 | 0.015 | 0.014 | 0.013 | 0.014 | 0.019 | 0.015 | 0.010 | 0.016 | ||
Colony 1, Replicate 3 | 2.06E+02 | 5.10E+03 | 1.24E+04 | 4.32E+03 | -8.72E+02 | 1.26E+04 | 1.06E+04 | 2.55E+03 | 3.15 | 71.60 | 142.94 | 82.81 | -13.36 | 224.95 | 94.95 | 35.84 | 0.012 | 0.011 | 0.009 | 0.015 | 0.012 | 0.014 | 0.007 | 0.011 | ||
Colony 1, Replicate 4 | 1.54E+02 | 3.95E+03 | 9.87E+03 | 4.22E+03 | -4.00E+02 | 4.57E+03 | 6.63E+03 | 2.07E+03 | 3.34 | 75.65 | 188.93 | 86.16 | -8.18 | 251.10 | 93.09 | 39.68 | 0.017 | 0.015 | 0.015 | 0.016 | 0.016 | 0.043 | 0.011 | 0.015 | ||
Colony 2, Replicate 1 | 3.59E+02 | 3.23E+03 | 6.90E+03 | 6.33E+03 | -5.53E+00 | 1.27E+04 | 8.16E+03 | 2.34E+03 | 6.42 | 57.80 | 167.26 | 121.24 | -0.12 | 210.56 | 125.02 | 23.92 | 0.014 | 0.014 | 0.019 | 0.015 | 0.017 | 0.013 | 0.012 | 0.008 | ||
Colony 2, Replicate 2 | 2.61E+02 | 2.50E+03 | 8.23E+03 | 5.76E+03 | 5.22E+00 | 2.06E+03 | 7.83E+03 | 1.27E+03 | 8.68 | 67.04 | 126.04 | 117.65 | 0.10 | 160.67 | 109.96 | 25.97 | 0.026 | 0.021 | 0.012 | 0.016 | 0.015 | 0.061 | 0.011 | 0.016 | ||
Colony 2, Replicate 3 | -1.10E+02 | 2.06E+03 | 3.32E+03 | 6.54E+03 | -3.02E+02 | 1.03E+04 | 7.15E+03 | 9.63E+02 | -2.24 | 52.48 | 165.10 | 125.30 | -5.79 | 171.15 | 100.39 | 17.21 | 0.016 | 0.020 | 0.039 | 0.015 | 0.015 | 0.013 | 0.011 | 0.014 | ||
Colony 2, Replicate 4 | 6.05E+02 | 3.65E+03 | 1.09E+04 | 6.43E+03 | -6.19E+02 | 1.25E+04 | 7.84E+03 | 1.07E+03 | 10.04 | 65.19 | 125.37 | 139.54 | -12.64 | 175.89 | 130.15 | 20.51 | 0.013 | 0.014 | 0.009 | 0.017 | 0.016 | 0.011 | 0.013 | 0.015 | ||
Hour 6: | Neg. Contr | Pos. Contr | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | Neg. Contr | Pos. Contr | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | Neg. Contr | Pos. Contr | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | ||
Colony 1, Replicate 1 | 6.47E+01 | 4.92E+03 | 1.39E+04 | 4.02E+03 | 8.51E+01 | 2.04E+04 | 6.02E+03 | 1.32E+03 | 35.83 | 1974.25 | 354.18 | 2247.35 | 43.88 | 677.19 | 184.55 | 535.17 | 0.434 | 0.314 | 0.020 | 0.438 | 0.404 | 0.026 | 0.024 | 0.318 | ||
Colony 1, Replicate 2 | 7.65E+01 | 6.08E+03 | 2.18E+04 | 3.88E+03 | 1.11E+02 | 2.37E+04 | 1.77E+04 | 1.46E+03 | 38.60 | 1970.33 | 389.12 | 2180.23 | 56.32 | 756.55 | 202.83 | 567.00 | 0.395 | 0.254 | 0.014 | 0.440 | 0.399 | 0.025 | 0.009 | 0.304 | ||
Colony 1, Replicate 3 | 8.71E+01 | 5.98E+03 | 2.19E+04 | 4.34E+03 | 8.33E+01 | 2.31E+04 | 1.24E+04 | 1.40E+03 | 46.49 | 2062.39 | 335.74 | 1768.69 | 33.30 | 767.86 | 189.68 | 551.31 | 0.418 | 0.270 | 0.012 | 0.319 | 0.313 | 0.026 | 0.012 | 0.308 | ||
Colony 1, Replicate 4 | 8.98E+01 | 6.58E+03 | 4.92E+04 | 4.33E+03 | 1.05E+02 | 2.93E+04 | 2.62E+04 | 1.53E+03 | 46.34 | 2057.54 | 313.85 | 2101.34 | 51.96 | 749.16 | 234.18 | 673.15 | 0.404 | 0.245 | 0.005 | 0.380 | 0.387 | 0.020 | 0.007 | 0.344 | ||
Colony 2, Replicate 1 | 8.80E+01 | 2.73E+03 | 1.46E+04 | 4.82E+03 | 1.16E+02 | 1.33E+04 | 9.85E+03 | 1.47E+03 | 48.77 | 1237.42 | 242.44 | 2159.12 | 67.76 | 389.75 | 364.88 | 675.30 | 0.434 | 0.355 | 0.013 | 0.351 | 0.456 | 0.023 | 0.029 | 0.361 | ||
Colony 2, Replicate 2 | 5.50E+01 | 3.54E+03 | 1.47E+04 | 3.67E+03 | 8.10E+01 | 1.95E+04 | 1.05E+04 | 1.39E+03 | 34.21 | 1501.41 | 300.03 | 2115.11 | 51.51 | 397.99 | 444.01 | 843.54 | 0.487 | 0.332 | 0.016 | 0.451 | 0.498 | 0.016 | 0.033 | 0.474 | ||
Colony 2, Replicate 3 | 5.28E+01 | 2.98E+03 | 1.37E+04 | 4.88E+03 | 8.68E+01 | 1.75E+04 | 9.50E+03 | 1.41E+03 | 32.21 | 1611.62 | 296.84 | 2598.62 | 53.55 | 379.00 | 400.38 | 850.63 | 0.478 | 0.424 | 0.017 | 0.417 | 0.483 | 0.017 | 0.033 | 0.471 | ||
Colony 2, Replicate 4 | 8.51E+01 | 2.90E+03 | 1.18E+04 | 4.87E+03 | 7.92E+01 | 1.53E+04 | 1.07E+04 | 1.60E+03 | 51.50 | 1602.12 | 256.74 | 2832.42 | 49.36 | 351.94 | 450.70 | 905.72 | 0.474 | 0.432 | 0.017 | 0.456 | 0.488 | 0.018 | 0.033 | 0.442 |
Colony Forming Unit Data and Calculations
Number of Colonies Found on Plates | ||||
Sample | 10^4 Dilution | 10^5 Dilution | 10^6 Dilution | CFU Calculations |
1.1 | 285 | 31 | 3 | 2.28E+07 |
1.2 | 276 | 34 | 2 | 2.21E+07 |
1.3 | 188 | 39 | 3 | 1.50E+07 |
2.1 | 282 | 62 | 11 | 2.26E+07 |
2.2 | TNTC | 30 | 11 | 2.40E+07 |
2.3 | TNTC | 106 | 4 | 8.48E+07 |
3.1 | TNTC | 34 | 2 | 2.72E+07 |
3.2 | TNTC | 116 | 2 | 9.28E+07 |
3.3 | 152 | 3 | 0 | 1.22E+07 |
4.1 | TNTC | 42 | 6 | 3.36E+07 |
4.2 | TNTC | 38 | 6 | 3.04E+07 |
4.3 | TNTC | 46 | 17 | 3.68E+07 |
TNTC stands for “too numerous to count” and represents any dilution plate with over 300 colonies.
References
- iGEM 2018 Interlab Study Protocol
- https://2018.igem.org/Measurement/InterLab