Erwinions
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?
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.
The InterLab Study has three main components: the calibration, the cell measurement and the colony forming units per 0.1 OD600
Interlab Results: Part I
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.
OD600 Reference Point
LUDOX CL-X
H2 O
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
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
Particle Standard Curve (log scale)
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
Fluorescein Standard Curve (log scale)
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 ="T oo N umerous T o C ount"
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 ="T oo N umerous T o C ount"