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.

  1. Comparison of OD600 with a known concentration of beads
  2. 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<sub>600</sub> at 0 hrs
Absorbance<sub>600</sub> at 6 hrs

Absorbance at 600nm recorded 0hrs (left) and 6hrs (right) after inoculation.


Fluorescence graphs

Fluorescence at 0 hrs
Fluorescence at 6 hrs

Fluorescence at 485nm excitation, 525nm emission - recorded at 0hrs (left) and 6hrs (right) after inoculation.


MEFL/Particle graphs

MEFL/particle for 0hrs
MEFL/particle for 0hrs

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.