Team:Purdue/InterLab

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Interlab

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

See iGEM 2018 InterLab Study Protocol

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. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 LB + Chlor (blank) Hour 0: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 LB + Chlor (blank)
Colony 1, Replicate 1 3.8675 78.0275 175.3375 96.3575 -6.7025 219.2175 121.7075 40.9775 0.3275 Colony 1, Replicate 1 0.01725 0.02625 0.01625 0.01625 0.01725 0.01425 0.01125 0.01525 0.00025
Colony 1, Replicate 2 6.8075 69.7375 156.0075 135.6975 -6.5625 219.7075 111.1175 40.6875 -2.6225 Colony 1, Replicate 2 0.01525 0.01425 0.01325 0.01425 0.01925 0.01525 0.01025 0.01625 0.00025
Colony 1, Replicate 3 4.4175 72.8675 144.2075 84.0775 -12.0925 226.2175 96.2175 37.1075 1.2675 Colony 1, Replicate 3 0.01425 0.01325 0.01125 0.01725 0.01425 0.01625 0.00925 0.01325 0.00225
Colony 1, Replicate 4 5.3375 77.6475 190.9275 88.1575 -6.1825 253.0975 95.0875 41.6775 1.9975 Colony 1, Replicate 4 0.01625 0.01425 0.01425 0.01525 0.01525 0.04225 0.01025 0.01425 -0.00075
Colony 2, Replicate 1 5.7075 57.0875 166.5475 120.5275 -0.8325 209.8475 124.3075 23.2075 -0.7125 Colony 2, Replicate 1 0.01425 0.01425 0.01925 0.01525 0.01725 0.01325 0.01225 0.00825 0.00025
Colony 2, Replicate 2 6.4375 64.7975 123.7975 115.4075 -2.1425 158.4275 107.7175 23.7275 -2.2425 Colony 2, Replicate 2 0.02525 0.02025 0.01125 0.01525 0.01425 0.06025 0.01025 0.01525 -0.00075
Colony 2, Replicate 3 0.0075 54.7275 167.3475 127.5475 -3.5425 173.3975 102.6375 19.4575 2.2475 Colony 2, Replicate 3 0.01525 0.01925 0.03825 0.01425 0.01425 0.01225 0.01025 0.01325 -0.00075
Colony 2, Replicate 4 9.7775 64.9275 125.1075 139.2775 -12.9025 175.6275 129.8875 20.2475 -0.2625 Colony 2, Replicate 4 0.01225 0.01325 0.00825 0.01625 0.01525 0.01025 0.01225 0.01425 -0.00075
Hour 6: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 LB + Chlor (blank) Hour 6: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 LB + Chlor (blank)
Colony 1, Replicate 1 45.4775 1983.898 363.8275 2256.998 53.5275 686.8375 194.1975 544.8175 9.6475 Colony 1, Replicate 1 0.43375 0.31375 0.01975 0.43775 0.40375 0.02575 0.02375 0.31775 -0.00025
Colony 1, Replicate 2 27.2675 1958.998 377.7875 2168.898 44.9875 745.2175 191.4975 555.6675 -11.3325 Colony 1, Replicate 2 0.39775 0.25675 0.01675 0.44275 0.40175 0.02775 0.01175 0.30675 0.00275
Colony 1, Replicate 3 38.7975 2054.698 328.0475 1760.998 25.6075 760.1675 181.9875 543.6175 -7.6925 Colony 1, Replicate 3 0.42175 0.27375 0.01575 0.32275 0.31675 0.02975 0.01575 0.31175 0.00375
Colony 1, Replicate 4 40.4975 2051.698 308.0075 2095.498 46.1175 743.3175 228.3375 667.3075 -5.8425 Colony 1, Replicate 4 0.41375 0.25475 0.01475 0.38975 0.39675 0.02975 0.01675 0.35375 0.00975
Colony 2, Replicate 1 46.5475 1235.198 240.2175 2156.898 65.5375 387.5275 362.6575 673.0775 -2.2225 Colony 2, Replicate 1 0.43075 0.35175 0.00975 0.34775 0.45275 0.01975 0.02575 0.35775 -0.00325
Colony 2, Replicate 2 43.6975 1510.898 309.5175 2124.598 60.9975 407.4775 453.4975 853.0275 9.4875 Colony 2, Replicate 2 0.48375 0.32875 0.01275 0.44775 0.49475 0.01275 0.02975 0.47075 -0.00325
Colony 2, Replicate 3 38.0875 1617.498 302.7175 2604.498 59.4275 384.8775 406.2575 856.5075 5.8775 Colony 2, Replicate 3 0.47375 0.41975 0.01275 0.41275 0.47875 0.01275 0.02875 0.46675 -0.00425
Colony 2, Replicate 4 53.5775 1604.198 258.8175 2834.498 51.4375 354.0175 452.7775 907.7975 2.0775 Colony 2, Replicate 4 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. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 Neg. Control Pos. Control 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. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 Neg. Control Pos. Control 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. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 Neg. Control Pos. Control 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. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 Neg. Control Pos. Control 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

  1. iGEM 2018 Interlab Study Protocol
  2. https://2018.igem.org/Measurement/InterLab

Special Thanks

to all those that made this possible

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