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Revision as of 01:10, 25 September 2018
![](https://static.igem.org/mediawiki/2018/a/ab/T--Tec-Chihuahua--interlabLetters.png)
All of the 2018 iGEM teams were invited to participate in the Fifth International InterLaboratory Measurement Study in synthetic biology. Team Tec-Chihuahua is excited about working on an international and collaborative experiment!
The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements. In order to address this challenge, the iGEM committee provides researchers with detailed protocols and requires data analysis on the measurement of GFP. This year, the million dollar question 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?
For more information, check out iGEM's official InterLab Study webpage. Below are the protocols we used and the data that we collected for the InterLab Study.
Calibration Measurements
OD600 Reference Point
The first calibration was to obtain a conversion factor to transform the absorbance data from the plate reader into a comparable OD 600 measurement, using LUDOX CL-X as a single point reference.
LUDOX CL-X | H2O | |
---|---|---|
Replicate 1 | 0.054 | 0.04 |
Replicate 2 | 0.051 | 0.035 |
Replicate 3 | 0.051 | 0.035 |
Replicate 4 | 0.052 | 0.035 |
Arith. Mean | 0.052 | 0.036 |
Corrected Abs600 | 0.016 | |
Reference OD600 | 0.063 | |
OD600/Abs600 | 4 |
Particle Standard Curve
In the second calibration was obtained a standard curve of particle concentration which was used to convert Abs 600 measurements to an estimated number of cells. It was measured a dilution of monodisperse silica microspheres.
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 | 0.601 | 0.236 | 0.171 | 0.105 | 0.071 | 0.053 | 0.045 | 0.039 | 0.038 | 0.037 | 0.036 | 0.035 |
Replicate 2 | 0.49 | 0.263 | 0.156 | 0.102 | 0.068 | 0.052 | 0.044 | 0.041 | 0.039 | 0.036 | 0.034 | 0.037 |
Replicate 3 | 0.519 | 0.269 | 0.191 | 0.098 | 0.07 | 0.051 | 0.043 | 0.04 | 0.037 | 0.039 | 0.037 | 0.034 |
Replicate 4 | 0.516 | 0.269 | 0.171 | 0.102 | 0.071 | 0.054 | 0.044 | 0.039 | 0.039 | 0.036 | 0.035 | 0.034 |
Arith. Mean | 0.532 | 0.259 | 0.172 | 0.102 | 0.07 | 0.053 | 0.044 | 0.04 | 0.038 | 0.037 | 0.036 | 0.035 |
Arith. Std Dev. | 0.048 | 0.016 | 0.014 | 0.003 | 0.001 | 0.001 | 0.001 | 0.001 | 0.001 | 0.001 | 0.001 | 0.001 |
Arith. Net Mean | 0.497 | 0.224 | 0.137 | 0.067 | 0.035 | 0.018 | 0.009 | 0.005 | 0.003 | 0.002 | 0.001 |
Particle Standard Curve
![](https://static.igem.org/mediawiki/2018/6/65/T--Tec-Chihuahua--Calibracion_2.1.png)
Particle Standard Curve (log scale)
![](https://static.igem.org/mediawiki/2018/c/c3/T--Tec-Chihuahua--Calibracion_2.2.png)
Particles/OD600 | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
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 |
Mean Particles/Abs600 | 4.74E+08 | 5.25E+08 | 4.29E+08 | 4.41E+08 | 4.20E+08 | 4.20E+08 | 4.08E+08 | 3.87E+08 | 2.83E+08 | 2.30E+08 | 4.60E+08 |
Mean of med-high levels | 4.47E+08 |
Fluorescence Standard Curve
Fluorescein uM | 10 | 5 | 2.5 | 1.25 | 0.625 | 0.313 | 0.156 | 0.078 | 0.039 | 0.0195 | 0.0098 | 0 |
---|---|---|---|---|---|---|---|---|---|---|---|---|
Replicate 1 | 1852 | 969.7 | 538.3 | 275.5 | 136.1 | 71.11 | 35.16 | 17.74 | 8.998 | 4.618 | 2.761 | 0.3263 |
Replicate 2 | 1929 | 956.8 | 523.7 | 284.7 | 132.6 | 66.21 | 36.66 | 17.95 | 8.892 | 4.856 | 2.553 | 0.34 |
Replicate 3 | 1922 | 977.1 | 516.5 | 281.1 | 150.3 | 75.65 | 40.22 | 19.54 | 10.57 | 5.299 | 2.931 | 0.3541 |
Replicate 4 | 1929 | 980.4 | 518.8 | 277.4 | 144.7 | 72.36 | 36.64 | 18.28 | 9.432 | 4.724 | 2.5 | 0.3667 |
Arith. Mean | 1.91E+03 | 9.71E+02 | 5.24E+02 | 2.80E+02 | 1.41E+02 | 7.13E+01 | 3.72E+01 | 1.84E+01 | 9.47E+00 | 4.87E+00 | 2.69E+00 | 3.47E-01 |
Arith. Std. Dev. | 3.75E+01 | 1.05E+01 | 9.79E+00 | 4.08E+00 | 8.06E+00 | 3.92E+00 | 2.15E+00 | 8.06E-01 | 7.68E-01 | 2.99E-01 | 1.98E-01 | 1.75E-02 |
Arith. Net Mean | 1.91E+03 | 9.71E+02 | 5.24E+02 | 2.79E+02 | 1.41E+02 | 7.10E+01 | 3.68E+01 | 1.80E+01 | 9.13E+00 | 4.53E+00 | 2.34E+00 |
Fluorescein Standard Curve
![](https://static.igem.org/mediawiki/2018/b/b5/T--Tec-Chihuahua--Calibracion_3.1.png)
Fluorescein Standard Curve (log scale)
![](https://static.igem.org/mediawiki/2018/0/05/T--Tec-Chihuahua--Calibracion_3.2.png)
Fluorescein uM | 10 | 5 | 2.5 | 1.25 | 0.63 | 0.31 | 0.16 | 0.08 | 0.04 | 0.02 | 0.01 |
---|---|---|---|---|---|---|---|---|---|---|---|
uM Fluorescein/a.u. | 5.24E-03 | 5.15E-03 | 4.77E-03 | 4.48E-03 | 4.45E-03 | 4.40E-03 | 4.24E-03 | 4.33E-03 | 4.28E-03 | 4.31E-03 | 4.17E-03 |
Mean uM Fluorescein/a.u. | 4.65E-03 | ||||||||||
MEFL/a.u. | 2.80E+10 |
Interlab Results: Part I
Fluorescence Raw Readings
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 | 5.482 | 8.456 | 18.54 | 9.417 | 5.513 | 18.04 | 26.03 | 6.729 | 4.351 |
Colony 1, Replicate 2 | 4.915 | 8.344 | 18.3 | 9.125 | 5.328 | 17.97 | 25.61 | 6.249 | 4.244 |
Colony 1, Replicate 3 | 5.244 | 8.183 | 18.5 | 9.04v | 5.163 | 18.2v | 25.89 | 6.73 | 4.447 |
Colony 1, Replicate 4 | 5.426 | 8.369 | 17.98 | 8.748 | 5.434 | 17.18 | 25.73 | 6.637 | 4.321 |
Colony 2, Replicate 1 | 5.348 | 7.593 | 20.28 | 8.4 | 5.534 | 17.25 | 22.39 | 5.868 | 4.355 |
Colony 2, Replicate 2 | 5.211 | 7.304 | 18.93 | 8.121 | 5.333 | 16.32 | 21.94 | 5.796 | 4.402 |
Colony 2, Replicate 3 | 5.308 | 7.355 | 19.89 | 8.181 | 5.408 | 16.89 | 21.81 | 5.982 | 4.399 |
Colony 2, Replicate 4 | 5.376 | 7.487 | 19.38 | 8.325 | 5.272 | 16.78 | 21.2 | 5.992 | 4.458 |
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 | 5.216 | 11.56 | 45.15 | 8.42 | 5.249 | 37.7 | 44.03 | 7.702 | 2.608 |
Colony 1, Replicate 2 | 5.1 | 12.65 | 45.74 | 8.949 | 5.008 | 38.49 | 47.11 | 7.273 | 3.704 |
Colony 1, Replicate 3 | 5.051 | 12.33 | 45.71 | 14.25 | 5.063 | 37.05 | 46.05 | 7.627 | 3.382 |
Colony 1, Replicate 4 | 4.967 | 12.71 | 45.11 | 14.56 | 5.018 | 39.27 | 45.37 | 7.444 | 4.121 |
Colony 2, Replicate 1 | 4.917 | 12 | 46.24 | 11.25 | 5.11 | 40.01 | 41.62 | 6.062 | 3.163 |
Colony 2, Replicate 2 | 4.896 | 12.41 | 47.24 | 10.74 | 5.003 | 38.93 | 42.12 | 6.935 | 3.178 |
Colony 2, Replicate 3 | 4.973 | 12.03 | 47.16 | 10.94 | 5.224 | 40.58 | 40.07 | 6.718 | 3.464 |
Colony 2, Replicate 4 | 4.867 | 12.54 | 46.62 | 10.87 | 5.001 | 40.69 | 38.35 | 6.513 | 3.5 |
Abs600 Raw Readings
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 | 0.074 | 0.076 | 0.07 | 0.077 | 0.08 | 0.085 | 0.073 | 0.072 | 0.039 |
Colony 1, Replicate 2 | 0.073 | 0.079 | 0.07 | 0.072 | 0.073 | 0.075 | 0.073 | 0.074 | 0.041 |
Colony 1, Replicate 3 | 0.079 | 0.078 | 0.067 | 0.076 | 0.074 | 0.075 | 0.069 | 0.078 | 0.04 |
Colony 1, Replicate 4 | 0.077 | 0.082 | 0.073 | 0.077 | 0.079 | 0.072 | 0.071 | 0.077 | 0.04 |
Colony 2, Replicate 1 | 0.079 | 0.079 | 0.077 | 0.073 | 0.074 | 0.076 | 0.072 | 0.076 | 0.04 |
Colony 2, Replicate 2 | 0.079 | 0.075 | 0.072 | 0.07 | 0.071 | 0.069 | 0.07 | 0.071 | 0.04 |
Colony 2, Replicate 3 | 0.075 | 0.076 | 0.075 | 0.071 | 0.081 | 0.072 | 0.068 | 0.073 | 0.04 |
Colony 2, Replicate 4 | 0.074 | 0.076 | 0.073 | 0.067 | 0.073 | 0.076 | 0.093 | 0.073 | 0.039 |
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 | 0.176 | 0.155 | 0.187 | 0.113 | 0.177 | 0.16 | 0.122 | 0.152 | 0.037 |
Colony 1, Replicate 2 | 0.168 | 0.164 | 0.174 | 0.122 | 0.18 | 0.174 | 0.134 | 0.155 | 0.038 |
Colony 1, Replicate 3 | 0.169 | 0.154 | 0.166 | 0.174 | 0.182 | 0.161 | 0.129 | 0.154 | 0.037 |
Colony 1, Replicate 4 | 0.169 | 0.147 | 0.171 | 0.171 | 0.178 | 0.166 | 0.122 | 0.154 | 0.038 |
Colony 2, Replicate 1 | 0.172 | 0.175 | 0.173 | 0.163 | 0.18 | 0.185 | 0.129 | 0.136 | 0.038 |
Colony 2, Replicate 2 | 0.173 | 0.163 | 0.184 | 0.153 | 0.171 | 0.195 | 0.121 | 0.155 | 0.038 |
Colony 2, Replicate 3 | 0.178 | 0.165 | 0.178 | 0.166 | 0.185 | 0.208 | 0.132 | 0.148 | 0.038 |
Colony 2, Replicate 4 | 0.159 | 0.159 | 0.184 | 0.155 | 0.174 | 0.172 | 0.117 | 0.142 | 0.037 |
Part II: Colony Forming Units Results
Colony Counting for Dilution Factors Plates
Culture 1 | Culture 2 | ||||||
Replicate 1 | Replicate 2 | Replicate 3 | Replicate 1 | Replicate 2 | Replicate 3 | ||
Positive Control | 8.00E+04 | TNTC | TNTC | TNTC | TNTC | TNTC | TNTC |
8.00E+05 | 273 | TNTC | TNTC | 69 | 84 | 99 | |
8.00E+04 | 17 | TNTC | 41 | 9 | 35 | 20 | |
Negative Control | 8.00E+04 | TNTC | TNTC | TNTC | TNTC | TNTC | TNTC |
8.00E+05 | TNTC | TNTC | TNTC | TNTC | 283 | 300 | |
8.00E+04 | TNTC | 59 | 240 | 72 | 33 | 27 |
TNTC="Too Numerous To Count"
Dilution OD600 Measurements
Culture 1 | Culture 2 | |||||
Replicate 1 | Replicate 2 | Replicate 3 | Replicate 1 | Replicate 2 | Replicate 3 | |
Positive Control | 0.099 | 0.105 | 0.102 | 0.097 | 0.100 | 0.096 |
Negative Control | 0.109 | 0.103 | 0.105 | 0.101 | 0.101 | 0.099 |
CFU Calculations
Culture 1 | Culture 2 | |||||
Replicate 1 | Replicate 2 | Replicate 3 | Replicate 1 | Replicate 2 | Replicate 3 | |
Positive Control | 1.36E+08 | TNTC | 3.28E+08 | 5.52E+07 | 6.72E+07 | 7.92E+07 |
Negative Control | TNTC | 4.72E+08 | 1.92E+09 | 5.76E+08 | 2.64E+08 | 2.16E+08 |
TNTC="Too Numerous To Count"