Difference between revisions of "Team:Tec-Chihuahua/InterLab"

Line 1,119: Line 1,119:
 
   width: 100%;
 
   width: 100%;
 
   padding-left: 175px;
 
   padding-left: 175px;
−
   padding-right: 175px;
+
   padding-right: 225px;
 
}
 
}
  

Revision as of 01:11, 25 September 2018

Erwinions





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.

Competent Cells Protocol

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)

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
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


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"

Follow us on
social media:


Instagram Facebook Twitter Snapchat Gmail

We thank our
sponsors:


Thermo Neb IDT Snap QTEc ITESM