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
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 | 0.297 | 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 appreciate Prof. Shi Hu for permitting us to use the plate reader from his lab. Besides, we want to thank Postgraduate instructor Tian Li for teaching us how to use a plate reader.