Team:IISc-Bangalore/InterLab
InterLab
Introduction
Fluorescence is a parameter that accurately correlates with the amount of the agent producing it. In case of proteins, the fluorescence of GFP is something that can be measured with a very high precision (limited only by half life and folding times). However, the quantification and comparison of fluorescence across multiple labs is a difficult task because of the difference in the way these measurements are reported. The goal of the fifth InterLab study is to reduce the variability in the fluorescence measurements reported across labs. The idea is to use the cell count (instead of the OD600) as a quantity using which the fluorescence is normalised to an absolute value.
As per iGEM, the big question we are answering is:
“Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?”
The aggregate fluorescence itself is reported in terms of Molecules of Equivalent Fluorescein (MEFL) which is then divided by the number of cells in the sample obtained using two methods.
- Comparison of OD600 with a known concentration of beads
- CFU counting
Calibration 1
Abs600 to OD600 conversion - LUDOX Protocol
LUDOX CL-X - 45% colloidal silica suspension - was used to obtain a conversion factor of the Abs600 (Measured at our lab) reading obtained from the plate reader into the equivalent OD600 reading (Measured by iGEM). This was used in the latter parts of the InterLab study to correlate the OD600 measurements to the number of cells in a sample. Measurement of Abs600 can be done in parallel in a high-throughput manner because of the presence of plate readers that can do so. Hence, this calibration is integral in ensuring fast and accurate reporting of ODs.
| LUDOX CL-X | H2O | |
|---|---|---|
| Replicate 1 | 0.0661 | 0.0516 |
| Replicate 2 | 0.0692 | 0.0504 |
| Replicate 3 | 0.0646 | 0.0502 |
| Replicate 4 | 0.0693 | 0.0498 |
| Mean | 0.067 | 0.051 |
| Corrected Abs600 | 0.017 | |
| Reference OD600 | 0.063 | |
| OD600/Abs600 | 3.756 |
Calibration 2
Abs600 to particle concentration conversion - Particle standard curve
Dilutions of silica microspheres from the stock provided by iGEM were made in quadruplicates and the Abs600 was measured using the plate reader. The curve obtained from this calibration provides an estimate of the number of cells in a sample with given Abs600 because of the roughly similar sizes of E. coli cells and the silica beads used in the experiment.
| Number of Particles | 2.35E+08 | 1.18E+08 | 5.88E+07 | 2.94E+07 | 1.47E+07 | 7.35E+06 | 3.68E+06 | 1.84E+06 | 9.19E+05 | 4.60E+05 | 2.30E+05 | 0 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Replicate 1 | 7.131E-01 | 4.167E-01 | 2.528E-01 | 1.431E-01 | 1.122E-01 | 8.78E-02 | 6.28E-02 | 5.61E-02 | 5.14E-02 | 5.07E-02 | 5.22E-02 | 4.92E-02 |
| Replicate 2 | 6.966E-01 | 4.11E-01 | 2.509E-01 | 1.564E-01 | 1.047E-01 | 7.62E-02 | 5.34E-02 | 4.53E-02 | 4.16E-02 | 4.31E-02 | 4.23E-02 | 4.79E-02 |
| Replicate 3 | 7.073E-01 | 4.866E-01 | 2.087E-01 | 1.528E-01 | 1.114E-01 | 8.32E-02 | 6.E-02 | 5.21E-02 | 4.95E-02 | 5.04E-02 | 4.55E-02 | 4.68E-02 |
| Replicate 4 | 7.625E-01 | 3.812E-01 | 2.615E-01 | 1.506E-01 | 1.09E-01 | 8.4E-02 | 6.65E-02 | 5.17E-02 | 4.65E-02 | 5.28E-02 | 4.7E-02 | 4.41E-02 |
| Arith. Mean | 0.720 | 0.424 | 0.243 | 0.151 | 0.109 | 0.083 | 0.061 | 0.051 | 0.047 | 0.049 | 0.047 | 0.047 |
| Arith. Std.Dev. | 0.029 | 0.045 | 0.024 | 0.006 | 0.003 | 0.005 | 0.006 | 0.004 | 0.004 | 0.004 | 0.004 | 0.002 |
| Arith. Net Mean | 0.673 | 0.377 | 0.196 | 0.104 | 0.062 | 0.036 | 0.014 | 0.004 | 0.000 | 0.002 | -0.000 |
| Number of Particles | 2.35E+08 | 1.18E+08 | 5.88E+07 | 2.94E+07 | 1.47E+07 | 7.35E+06 | 3.68E+06 | 1.84E+06 | 9.19E+05 | 4.60E+05 | 2.30E+05 | 0 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Mean particles / Abs600 | 3.50E+08 | 3.12E+08 | 2.99E+08 | 2.84E+08 | 2.36E+08 | 2.05E+08 | 2.69E+08 | 4.27E+08 | 3.68E+09 | 2.04E+08 | -9.19E+08 | 4.92E-02 |
Calibration 3
Relative fluoroscence to MEFL conversion - Fluoroscence standard curve
Dilutions of fluorescein from the stock provided by iGEM were made in quadruplicates and the fluorescence was measured using the plate reader. The standard curve of fluorescence for fluorescein concentration, which can be used for converting the cell-based readings into an equivalent fluorescein concentration, is as follows.
| Fluorescein uM | 10.00 | 5.00 | 2.50 | 1.25 | 0.63 | 0.31 | 0.16 | 0.08 | 0.04 | 0.02 | 0.01 | 0 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| uM Fluorescein/a.u. | 2.46E-04 | 2.07E-04 | 2.01E-04 | 1.96E-04 | 1.93E-04 | 1.93E-04 | 1.93E-04 | 1.92E-04 | 1.89E-04 | 1.90E-04 | 1.88E-04 | 4.92E-02 |
| Fluorescein uM | 10.00 | 5 | 2.5 | 1.25 | 0.625 | 0.313 | 0.156 | 0.078 | 0.039 | 0.0195 | 0.0098 | 0 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Replicate 1 | 44051 | 23662 | 11848 | 6424 | 3228 | 1602 | 800 | 424 | 216 | 109 | 55 | 4 |
| Replicate 2 | 43592 | 24013 | 12490 | 6176 | 3230 | 1592 | 805 | 388 | 215 | 101 | 57 | 4 |
| Replicate 3 | 43790 | 23654 | 12724 | 6451 | 3221 | 1634 | 815 | 413 | 198 | 107 | 55 | 4 |
| Replicate 4 | 31156 | 25354 | 12616 | 6513 | 3312 | 1663 | 840 | 416 | 212 | 110 | 56 | 3 |
| Arith. Mean | 4.065E+04 | 2.417E+04 | 1.242E+04 | 6.391E+03 | 3.248E+03 | 1.623E+03 | 8.150E+02 | 4.103E+02 | 2.103E+02 | 1.068E+02 | 5.575E+01 | 3.750E+00 |
| Arith. Std.Dev. | 6.330E+03 | 8.064E+02 | 3.928E+02 | 1.481E+02 | 4.301E+01 | 3.226E+01 | 1.780E+01 | 1.554E+01 | 8.342E+00 | 4.031E+00 | 9.574E-01 | 5.000E-01 |
| Arith. Net Mean | 4.064E+04 | 2.417E+04 | 1.242E+04 | 6.387E+03 | 3.244E+03 | 1.619E+03 | 8.113E+02 | 4.065E+02 | 2.065E+02 | 1.030E+02 | 5.200E+01 |
Cell Measurements
E.coli (DH5-alpha) was transformed with the plasmids given by iGEM. The colonies obtained were inoculated overnight. The net absorbance of the culture was then measured and diluted to 0.02 Abs6oo. After this, absorbance and fluorescence readings were taken for the sample at 0 hours and 6 hours.
Net Abs600
| 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 | 0.103 | 0.085 | 0.028 | 0.087 | 0.082 | 0.039 | 0.018 | 0.054 | 109 |
| Colony 1, Replicate 2 | 0.108 | 0.097 | 0.031 | 0.101 | 0.084 | 0.044 | 0.023 | 0.065 | 102 |
| Colony 1, Replicate 3 | 0.107 | 0.096 | 0.026 | 0.097 | 0.084 | 0.043 | 0.021 | 0.066 | 103 |
| Colony 1, Replicate 4 | 0.115 | 0.097 | 0.034 | 0.102 | 0.086 | 0.042 | 0.020 | 0.068 | 119 |
| Colony 2, Replicate 1 | 0.097 | 0.086 | 0.033 | 0.082 | 0.101 | 0.054 | 0.020 | 0.073 | 112 |
| Colony 2, Replicate 2 | 0.109 | 0.085 | 0.024 | 0.095 | 0.103 | 0.058 | 0.023 | 0.075 | 110 |
| Colony 2, Replicate 3 | 0.114 | 0.090 | 0.023 | 0.100 | 0.104 | 0.056 | 0.022 | 0.085 | 110 |
| Colony 2, Replicate 4 | 0.114 | 0.084 | 0.023 | 0.094 | 0.101 | 0.056 | 0.022 | 0.084 | 110 |
| 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 | 0.365 | 0.338 | 0.179 | 0.368 | 0.508 | 0.295 | 0.021 | 0.358 | 109 |
| Colony 1, Replicate 2 | 0.374 | 0.359 | 0.204 | 0.403 | 0.531 | 0.330 | 0.021 | 0.359 | 102 |
| Colony 1, Replicate 3 | 0.348 | 0.381 | 0.206 | 0.412 | 0.557 | 0.325 | 0.023 | 0.372 | 103 |
| Colony 1, Replicate 4 | 0.356 | 0.372 | 0.202 | 0.385 | 0.557 | 0.334 | 0.023 | 0.372 | 119 |
| Colony 2, Replicate 1 | 0.386 | 0.301 | 0.193 | 0.338 | 0.387 | 0.300 | 0.025 | 0.350 | 112 |
| Colony 2, Replicate 2 | 0.404 | 0.332 | 0.211 | 0.385 | 0.419 | 0.321 | 0.026 | 0.364 | 110 |
| Colony 2, Replicate 3 | 0.417 | 0.324 | 0.199 | 0.360 | 0.407 | 0.330 | 0.027 | 0.386 | 110 |
| Colony 2, Replicate 4 | 0.415 | 0.344 | 0.204 | 0.363 | 0.417 | 0.341 | 0.031 | 0.390 | 110 |
Net Fluorescein a.u.
| 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 | 8.00 | 405.00 | 1026.00 | 659.00 | 7.00 | 1095.00 | 801.00 | 137.00 | 109 |
| Colony 1, Replicate 2 | 20.00 | 436.00 | 1087.00 | 709.00 | 29.00 | 1162.00 | 852.00 | 160.00 | 102 |
| Colony 1, Replicate 3 | 23.00 | 449.00 | 1064.00 | 715.00 | 25.00 | 1220.00 | 854.00 | 182.00 | 103 |
| Colony 1, Replicate 4 | 9.00 | 433.00 | 1184.00 | 710.00 | 20.00 | 1181.00 | 843.00 | 169.00 | 119 |
| Colony 2, Replicate 1 | 0.00 | 462.00 | 891.00 | 583.00 | 10.00 | 1482.00 | 865.00 | 182.00 | 112 |
| Colony 2, Replicate 2 | 7.00 | 446.00 | 905.00 | 631.00 | 28.00 | 1557.00 | 968.00 | 184.00 | 110 |
| Colony 2, Replicate 3 | 15.00 | 497.00 | 874.00 | 633.00 | 16.00 | 1508.00 | 946.00 | 212.00 | 110 |
| Colony 2, Replicate 4 | 17.00 | 472.00 | 902.00 | 598.00 | 28.00 | 1494.00 | 935.00 | 204.00 | 110 |
| 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 | 21.00 | 920.00 | 3674.00 | 2325.00 | 54.00 | 6996.00 | 834.00 | 448.00 | 109 |
| Colony 1, Replicate 2 | 41.00 | 1009.00 | 4363.00 | 2616.00 | 72.00 | 7796.00 | 950.00 | 486.00 | 102 |
| Colony 1, Replicate 3 | 37.00 | 1054.00 | 4253.00 | 2633.00 | 73.00 | 7832.00 | 1009.00 | 518.00 | 103 |
| Colony 1, Replicate 4 | 23.00 | 1041.00 | 4154.00 | 2502.00 | 59.00 | 8005.00 | 986.00 | 510.00 | 119 |
| Colony 2, Replicate 1 | 27.00 | 1112.00 | 2641.00 | 1580.00 | 38.00 | 7690.00 | 1018.00 | 569.00 | 112 |
| Colony 2, Replicate 2 | 35.00 | 1215.00 | 2904.00 | 1761.00 | 58.00 | 8294.00 | 1032.00 | 589.00 | 110 |
| Colony 2, Replicate 3 | 34.00 | 1233.00 | 2809.00 | 1697.00 | 50.00 | 8525.00 | 1123.00 | 620.00 | 110 |
| Colony 2, Replicate 4 | 32.00 | 1277.00 | 2928.00 | 1721.00 | 55.00 | 8745.00 | 1148.00 | 640.00 | 110 |
Experimental Values : uM Fluorescein/OD
| 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 | 0.004 | 0.251 | 1.904 | 0.399 | 0.005 | 1.495 | 2.372 | 0.134 | 109 |
| Colony 1, Replicate 2 | 0.010 | 0.236 | 1.842 | 0.370 | 0.018 | 1.408 | 1.944 | 0.131 | 102 |
| Colony 1, Replicate 3 | 0.011 | 0.247 | 2.132 | 0.388 | 0.016 | 1.499 | 2.103 | 0.145 | 103 |
| Colony 1, Replicate 4 | 0.004 | 0.235 | 1.846 | 0.368 | 0.012 | 1.468 | 2.178 | 0.131 | 119 |
| Colony 2, Replicate 1 | 0.000 | 0.283 | 1.423 | 0.374 | 0.005 | 1.460 | 2.314 | 0.131 | 112 |
| Colony 2, Replicate 2 | 0.003 | 0.275 | 1.963 | 0.349 | 0.014 | 1.410 | 2.218 | 0.129 | 110 |
| Colony 2, Replicate 3 | 0.007 | 0.290 | 2.003 | 0.334 | 0.008 | 1.414 | 2.266 | 0.131 | 110 |
| Colony 2, Replicate 4 | 0.008 | 0.297 | 2.076 | 0.337 | 0.015 | 1.409 | 2.210 | 0.127 | 110 |
| 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 | 0.003 | 0.143 | 1.079 | 0.333 | 0.006 | 1.250 | 2.044 | 0.066 | 109 |
| Colony 1, Replicate 2 | 0.006 | 0.148 | 1.125 | 0.342 | 0.007 | 1.247 | 2.340 | 0.071 | 102 |
| Colony 1, Replicate 3 | 0.006 | 0.146 | 1.085 | 0.337 | 0.007 | 1.270 | 2.332 | 0.073 | 103 |
| Colony 1, Replicate 4 | 0.003 | 0.147 | 1.083 | 0.343 | 0.006 | 1.262 | 2.259 | 0.072 | 119 |
| Colony 2, Replicate 1 | 0.004 | 0.195 | 0.720 | 0.246 | 0.005 | 1.351 | 2.155 | 0.086 | 112 |
| Colony 2, Replicate 2 | 0.005 | 0.193 | 0.725 | 0.241 | 0.007 | 1.360 | 2.116 | 0.085 | 110 |
| Colony 2, Replicate 3 | 0.004 | 0.201 | 0.742 | 0.249 | 0.006 | 1.364 | 2.225 | 0.085 | 110 |
| Colony 2, Replicate 4 | 0.004 | 0.196 | 0.755 | 0.250 | 0.007 | 1.350 | 1.939 | 0.087 | 110 |
Experimental Values : MEFL/Particle
| 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 | 3.48E+02 | 2.12E+04 | 1.61E+05 | 3.37E+04 | 3.83E+02 | 1.27E+05 | 2.01E+05 | 1.14E+04 | 109 |
| Colony 1, Replicate 2 | 8.28E+02 | 2.00E+04 | 1.56E+05 | 3.13E+04 | 1.54E+03 | 1.19E+05 | 1.64E+05 | 1.10E+04 | 102 |
| Colony 1, Replicate 3 | 9.56E+02 | 2.09E+04 | 1.80E+05 | 3.29E+04 | 1.33E+03 | 1.27E+05 | 1.78E+05 | 1.23E+04 | 103 |
| Colony 1, Replicate 4 | 3.50E+02 | 1.98E+04 | 1.56E+05 | 3.11E+04 | 1.03E+03 | 1.24E+05 | 1.84E+05 | 1.11E+04 | 119 |
| Colony 2, Replicate 1 | 0.00E+00 | 2.39E+04 | 1.20E+05 | 3.17E+04 | 4.42E+02 | 1.24E+05 | 1.96E+05 | 1.11E+04 | 112 |
| Colony 2, Replicate 2 | 2.87E+02 | 2.33E+04 | 1.66E+05 | 2.96E+04 | 1.21E+03 | 1.19E+05 | 1.88E+05 | 1.09E+04 | 110 |
| Colony 2, Replicate 3 | 5.86E+02 | 2.46E+04 | 1.69E+05 | 2.83E+04 | 6.88E+02 | 1.20E+05 | 1.92E+05 | 1.11E+04 | 110 |
| Colony 2, Replicate 4 | 6.63E+02 | 2.51E+04 | 1.76E+05 | 2.85E+04 | 1.23E+03 | 1.19E+05 | 1.87E+05 | 1.08E+04 | 110 |
| 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 | 2.57E+02 | 1.21E+04 | 9.13E+04 | 2.82E+04 | 4.74E+02 | 1.06E+05 | 1.73E+05 | 5.59E+03 | 109 |
| Colony 1, Replicate 2 | 4.88E+02 | 1.25E+04 | 9.52E+04 | 2.89E+04 | 6.04E+02 | 1.05E+05 | 1.98E+05 | 6.04E+03 | 102 |
| Colony 1, Replicate 3 | 4.75E+02 | 1.23E+04 | 9.18E+04 | 2.85E+04 | 5.84E+02 | 1.07E+05 | 1.97E+05 | 6.21E+03 | 103 |
| Colony 1, Replicate 4 | 2.88E+02 | 1.25E+04 | 9.17E+04 | 2.90E+04 | 4.72E+02 | 1.07E+05 | 1.91E+05 | 6.11E+03 | 119 |
| Colony 2, Replicate 1 | 3.12E+02 | 1.65E+04 | 6.09E+04 | 2.08E+04 | 4.38E+02 | 1.14E+05 | 1.82E+05 | 7.26E+03 | 112 |
| Colony 2, Replicate 2 | 3.87E+02 | 1.63E+04 | 6.13E+04 | 2.04E+04 | 6.18E+02 | 1.15E+05 | 1.79E+05 | 7.22E+03 | 110 |
| Colony 2, Replicate 3 | 3.63E+02 | 1.70E+04 | 6.28E+04 | 2.11E+04 | 5.47E+02 | 1.15E+05 | 1.88E+05 | 7.17E+03 | 110 |
| Colony 2, Replicate 4 | 3.44E+02 | 1.66E+04 | 6.39E+04 | 2.11E+04 | 5.88E+02 | 1.14E+05 | 1.64E+05 | 7.32E+03 | 110 |
Absorbance graphs
Absorbance at 600nm recorded 0hrs (left) and 6hrs (right) after inoculation.
Fluorescence graphs
Fluorescence at 485nm excitation, 525nm emission - recorded at 0hrs (left) and 6hrs (right) after inoculation.
MEFL/Particle graphs
MEFL/Particle for the test devices recorded 0hrs (left) and 6hrs (right) after inoculation.
Colony Forming Units per 0.1 OD600 E.coli cultures
E.coli (DH5-alpha) was transformed with the plasmids of positive (BBa_I20270) and negative (R0040) controls given by iGEM. The colonies obtained were inoculated in 10ml tubes overnight. The culture was then diluted to 0.1 OD600. After a series of three 20-fold dilutions. Then the two more sets of sample cultures were prepared by performing two more 10- fold dilutions. These three sets of samples were then plated and the number of colonies obtained was counted.
| Colony | I20270 (1) | I20270 (2) | R0040 (1) | R0040 (2) | |
|---|---|---|---|---|---|
| Dilution | Replicate | No.of colonies | |||
| Dilution 3 | Replicate 1 | 207 |
TMTC | TMTC | TMTC |
| Replicate 2 | TMTC | TMTC | TMTC | 173 | |
| Replicate 3 | 147 | 269 | 294 | 152 | |
| Dilution 4 | Replicate 1 | 18 | 25 | 6 | 25 |
| Replicate 2 | 110 | 18 | 13 | 56 | |
| Replicate 3 | 54 | 14 | 18 | 29 | |
| Dilution 5 | Replicate 1 | 4 | 6 | 0 | 0 |
| Replicate 2 | 3 | 1 | 1 | 5 | |
| Replicate 3 | 1 | 0 | 1 | 0 | |
Conclusion
Test Device 5 was found to have the highest MEFL/Particle value of all the test devices. Test device 1 showed an interesting decreasing in MEFL/Particle value from 0hrs to 6hrs indicating a decrease in GFP expression while cell growth continued. The MEFL/Particle fluorescence obtained from Test Device 3 was also considerably high.
The colony count for both the positive and negative controls were found to be similar indicating no considerable effect of GFP expression on cell growth.