Interlab Study 2018
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?
Part I: Interlab Results
| |
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 |
|
| 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 |
|
Calibration 2.1
Calibration 2.2
Particles/OD
| 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 |
| 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 |
|
Calibration 3.1
Calibration 3.2
Fluorescein/a.u Measurements
| 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 |
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"
