Team:Tec-Chihuahua/InterLab

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 protocols were actually very simple to understand and we didn't have any problems following them. Without a doubt, it was an enlightening experience full of challenges that helped us improve laboratory work during the development of the project. When we started working on it, a small problem appeared since we only had the Top10 strain at our disposal. But the New England Biolabs sponsorship arrived just in time with the DH5α strain, which allowed us to properly undertake all the protocols.

Due to the fact that we didn't have a microplate reader, taking part in the InterLab Study was the perfect opportunity to learn how to use one. The only drawback we had was getting the equipment to take the measurements but, in the end, the Research Center for Advanced Materials (CIMAV) lent us their laboratories. We are very proud because this year was the first time Tec-Chihuahua successfully participated in the InterLab Study.

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 OD600 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.

Particle Standard Curve - Microsphere 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

In the third calibration, we generated a standard curve of fluorescence for fluorescein concentration, which was used to convert the cell-based readings to an equivalent fluorescein concentration.

Calibration 3: Fluorescence Standard Curve - Fluorescein Protocol
▼


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


Results Part I: Cell Measurement

After the calibrations protocols were done, we continued with the cell measurements which were made under the same condition. The results that were obtained are shown below.

Cell Measurement Protocol
▼

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


Results Part II: CFU per 0.1 OD600

Colony Forming Units per 0.1 OD600 E. coli Cultures Protocols
▼

Dilution OD600 Measurements

Thereafter, the protocol of colony forming units per 0.1 OD600 was made. The overnight cultures were diluted, obtaining the following results.

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


Colony Counting for Dilution Factors Plates

Following this, serial dilutions were made and the last three were spread on plate. The number of colonies after 19 hours of growth were:

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"


CFU Calculations

Finally, colony forming units (CFU) per 1mL of an OD600 = 0.1 culture were calculated. The results are as follows:

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