Team:SMMU-China/InterLab

Interlab

Introduction

Reliable and repeatable measurement is a key component of synthetic biology. However, it has been difficult to repeat the same measurements in different labs. There have been few opportunities to compare the fluorescence data because it has been reported in different units or because different groups process data in different ways.

With the aim to improve the measurement tools available to both the iGEM community and the synthetic biology community, the measurement committee invited all iGEM teams to participate in the fifth InterLab Study, which provides researchers with a detailed protocol and data analysis form, with the aim to produce common, comparable units for measuring GFP on different plate readers.

For a more detailed description visit the homepage of the iGEM's measurement committee. (https://2018.igem.org/Measurement/InterLab)

Device we received

Positive Control (BBa_I20270)
Negative Control (BBa_R0040)
Test Device1 (BBa_J364000)
Test Device2 (BBa_J364001)
Test Device3 (BBa_J364002)
Test Device4 (BBa_J364007)
Test Device5 (BBa_J364008)
Test Device6 (BBa_J364009)

Protocol

Strictly following the protocol provided by iGEM, we made three sets of unit calibration measurements: an OD600 reference point, a particle standard curve, and a fluorescein standard curve.

The transformation protocol(连接)

The interLab plate reader protocol（连接）

The raw data

LUDOX CL-X H20
Replicate 1 0.060 0.047
Replicate 2 0.059 0.045
Replicate 3 0.060 0.048
Replicate 4 0.064 0.056
Arith. Mean 0.061 0.049
Corrected Abs600 0.012
Reference OD600 0.063
OD600/Abs600 5.362

Stable 1. OD600Reference Point

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 0.528 0.315 0.163 0.126 0.104 0.076 0.064 0.047 0.04 0.041 0.043 0.043
Replicate 2 0.556 0.286 0.17 0.106 0.087 0.062 0.043 0.041 0.04 0.05 0.057 0.047
Replicate 3 0.524 0.289 0.161 0.109 0.104 0.058 0.051 0.048 0.036 0.049 0.053 0.042
Replicate 4 0.524 0.269 0.159 0.106 0.078 0.066 0.05 0.043 0.034 0.041 0.045 0.037
Arith. Mean 0.533 0.29 0.163 0.112 0.093 0.066 0.052 0.045 0.038 0.045 0.05 0.042
Arith. Std. Dev. 0.015 0.019 0.005 0.01 0.013 0.008 0.009 0.003 0.003 0.005 0.007 0.004
Arith. Net Mean 0.491 0.248 0.121 0.07 0.051 0.023 0.01 0.003 -0.005 0.003 0.007

Stable 2. Particle Count

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 18743 9954 5246 2864 1544 755 411 214 118 67 38 13
Replicate 2 19324 9825 5512 2915 1515 727 364 209 110 60 35 15
Replicate 3 19601 10382 5581 2870 1440 701 363 192 109 56 36 14
Replicate 4 19104 10615 5662 2816 1464 707 401 185 97 55 39 14
Arith. Mean 19190.00 10190.00 5500.00 2866.00 1491.00 722.50 384.80 200.00 108.50 59.50 37.00 14.00
Arith.Std.Dev. 362.40 368.00 180.20 40.50 47.31 24.35 24.88 13.74 8.66 5.45 1.83 0.82
Arith. Net Mean 19180.00 10180.00 5486.00 2852.00 1477.00 708.50 370.80 186.00 94.50 45.50 23.00

Stable 3. Fluorescein standard curve

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 0.297 0.227 0.126 0.231 0.186 0.141 0.152 0.221 0.117
Colony 1, Replicate 2 0.282 0.213 0.102 0.167 0.147 0.111 0.109 0.182 0.066
Colony 1, Replicate 3 0.261 0.222 0.104 0.164 0.146 0.112 0.103 0.206 0.041
Colony 1, Replicate 4 0.254 0.061 0.102 0.158 0.145 0.105 0.104 0.178 0.036
Colony 2, Replicate 1 0.278 0.232 0.113 0.176 0.15 0.107 0.109 0.201 0.035
Colony 2, Replicate 2 0.299 0.221 0.105 0.178 0.151 0.113 0.107 0.221 0
Colony 2, Replicate 3 0.256 0.216 0.121 0.157 0.151 0.111 0.107 0.199 0.037
Colony 2, Replicate 4 0.267 0.217 0.118 0.161 0.149 0.109 0.11 0.212 0.041
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 0.48 0.471 0.495 0.663 0.542 0.52 0.321 0.566 0.041
Colony 1, Replicate 2 0.491 0.481 0.493 0.644 0.543 0.532 0.331 0.574 0.046
Colony 1, Replicate 3 0.458 0.438 0.48 0.662 0.534 0.538 0.332 0.567 0.054
Colony 1, Replicate 4 0.523 0.535 0.536 0.701 0.588 0.563 0.361 0.599 0.064
Colony 2, Replicate 1 0.497 0.495 0.478 0.65 0.561 0.551 0.355 0.61 0.039
Colony 2, Replicate 2 0.512 0.486 0.512 0.665 0.563 0.549 0.354 0.612 0.037
Colony 2, Replicate 3 0.484 0.451 0.497 0.681 0.563 0.56 0.337 0.591 0.035
Colony 2, Replicate 4 0.478 0.501 0.49 0.676 0.569 0.552 0.347 0.201 0.036

Stable 4. Abs600 Raw Readings

Data analysis

Figure 1. Particle standard curve.

Figure 2.Fluorescein fluorescence standard curve.

Figure3 .The absorbance at 600nm over time. Samples were collected at 0 hour and 6 hours, and absorbance at 600nm were measured by the plate reader. Every device had two colonies.

Figure4 .The change of florescence over time. Samples were collected at 0 hour and 6hour and measured their fluorescence by the plate reader. Every device had two colonies.

Conclusion

We measured both fluorescence and absorbance of 2 biological replicates and 4 technical replicates. Abs600 values for device 5 are almost the lowest while the values of device 2 are almost .The colony 2 series are similar to colony1. Fluorescence value of device 1 is the highest while the value of device 3 is the lowest, which approaches to the value of counterpart of negative control.

Acknowledgement

We want to thank Postgraduate instructor Tian Li for teaching us how to use a plate reader.

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