Team:Jilin China/InterLab

Introduction

Reliable and repeatable measurement is a key component to all engineering disciplines. The same holds true for synthetic biology, which has also been called engineering biology. However, the ability to repeat measurements in different labs has been difficult. The Measurement Committee, through the InterLab study, has been developing a robust measurement procedure for green fluorescent protein (GFP) over the last three years. We chose GFP as the measurement marker for this study since it's one of the most used markers in synthetic biology and, as a result, most laboratories are equipped to measure this protein. We think it is a great chance for our team to make our own contribution to this project.

Device we received

  • Positive Control (BBa_I20270)
  • Negative Control (BBa_R0040)
  • Test Device 1 (BBa_J364000)
  • Test Device 2 (BBa_J364001)
  • Test Device 3 (BBa_J364002)
  • Test Device 4 (BBa_J364003)
  • Test Device 5 (BBa_J364004)
  • Test Device 6 (BBa_J364005)

Protocol

We strictly followed the protocol provided by iGEM. We measured the experiment of OD600 reference point and fitted the fluorescence standard curve first and then detected GFP expression in our E.coli with the plate reader Synergy HT from BioTek.

Result
OD600 reference point
LUDOX-HS40 H2O
Replicate 1 0.047 0.04
Replicate 2 0.045 0.04
Replicate 3 0.048 0.042
Replicate 4 0.048 0.041
Arith. Mean 0.047 0.04075
Corrected Abs600 0.00625
Reference OD600 0.0425
OD600/Abs600 6.8
Fluorescein standard curve
uM Fluorescein 50 25 12.5 6.25 3.125 1.5625 0.78125 0.39063 0.19531 0.097656 0.048828 0
Replicate 1 40649 26105 16044 8715 4461 2275 1197 594 298 160 76 4
Replicate 2 41111 26716 15422 8594 4540 2273 1170 593 300 152 77 4
Replicate 3 41157 26528 15611 8410 4472 2280 1159 629 277 145 76 5
Replicate 4 40760 26858 15662 8607 4392 2340 1174 591 297 157 80 4
Arith. Mean 40919.25 26551.75 15684.75 8581.5 4466.25 2292 1175 601.75 293 153.5 77.25 4.25
Arith. Std.Dev. 252.7771 327.0661 260.8031 126.5451 60.5881 32.1351 15.9792 18.2094 10.7393 6.5574 1.893 0.5






Raw Plate Reader Measurements
Dilution Calculation:



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 0.07 0.07 0.062 0.068 0.066 0.067 0.069 0.068 0.042
Colony 1, Replicate 2 0.07 0.068 0.063 0.063 0.063 0.065 0.069 0.065 0.041
Colony 1, Replicate 3 0.069 0.066 0.065 0.066 0.065 0.066 0.07 0.067 0.044
Colony 1, Replicate 4 0.072 0.069 0.065 0.069 0.067 0.067 0.07 0.067 0.044
Colony 2, Replicate 1 0.065 0.062 0.059 0.063 0.064 0.064 0.061 0.065 0.043
Colony 2, Replicate 2 0.064 0.061 0.058 0.063 0.065 0.062 0.062 0.063 0.044
Colony 2, Replicate 3 0.065 0.062 0.058 0.062 0.06 0.063 0.063 0.062 0.041
Colony 2, Replicate 4 0.065 0.063 0.059 0.063 0.066 0.063 0.063 0.065 0.044

Hour 2: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 LB + Chlor (blank)
Colony 1, Replicate 1 0.173 0.162 0.064 0.146 0.153 0.132 0.155 0.16 0.047
Colony 1, Replicate 2 0.188 0.164 0.07 0.15 0.158 0.133 0.167 0.157 0.044
Colony 1, Replicate 3 0.18 0.156 0.074 0.155 0.166 0.14 0.163 0.18 0.051
Colony 1, Replicate 4 0.181 0.162 0.073 0.155 0.167 0.136 0.172 0.184 0.045
Colony 2, Replicate 1 0.141 0.1 0.063 0.122 0.139 0.118 0.132 0.148 0.046
Colony 2, Replicate 2 0.15 0.101 0.065 0.122 0.141 0.121 0.132 0.149 0.043
Colony 2, Replicate 3 0.148 0.105 0.069 0.13 0.136 0.124 0.138 0.199 0.043
Colony 2, Replicate 4 0.148 0.116 0.066 0.137 0.147 0.12 0.14 0.153 0.044

Hour 4: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 LB + Chlor (blank)
Colony 1, Replicate 1 0.289 0.274 0.093 0.264 0.256 0.255 0.266 0.248 0.045
Colony 1, Replicate 2 0.297 0.282 0.098 0.272 0.255 0.264 0.275 0.274 0.042
Colony 1, Replicate 3 0.307 0.279 0.101 0.279 0.271 0.259 0.279 0.278 0.046
Colony 1, Replicate 4 0.289 0.297 0.104 0.281 0.263 0.26 0.285 0.283 0.044
Colony 2, Replicate 1 0.256 0.193 0.077 0.222 0.233 0.235 0.281 0.245 0.044
Colony 2, Replicate 2 0.269 0.192 0.083 0.239 0.242 0.238 0.295 0.249 0.044
Colony 2, Replicate 3 0.271 0.207 0.078 0.231 0.239 0.244 0.302 0.243 0.041
Colony 2, Replicate 4 0.267 0.192 0.082 0.229 0.247 0.237 0.293 0.257 0.045

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 0.358 0.337 0.168 0.351 0.318 0.328 0.332 0.32 0.044
Colony 1, Replicate 2 0.39 0.359 0.176 0.358 0.344 0.35 0.372 0.34 0.05
Colony 1, Replicate 3 0.399 0.36 0.177 0.368 0.347 0.346 0.359 0.343 0.045
Colony 1, Replicate 4 0.402 0.359 0.177 0.365 0.346 0.358 0.364 0.351 0.054
Colony 2, Replicate 1 0.315 0.277 0.183 0.322 0.301 0.348 0.33 0.32 0.047
Colony 2, Replicate 2 0.329 0.289 0.186 0.351 0.319 0.389 0.353 0.331 0.051
Colony 2, Replicate 3 0.339 0.301 0.189 0.358 0.344 0.371 0.359 0.362 0.052
Colony 2, Replicate 4 0.337 0.307 0.202 0.354 0.331 0.387 0.363 0.356 0.076



Abs600 Raw Readings




Conclusion

As our results showed, promoter J23101 was the strongest among the 3 promoters, which showed the highest GFP fluorescence. Promoter J23106 was moderate, and promoter J23117 was the lowest.

Acknowledgement

We really appreciated Prof. Yubin Ge for providing us the permission to use the plate reader from his lab. Besides, PhD candidate Cheng Hu taught us how to use a plate reader. BioTek service engineer Jeremy Gagne told us some settings of the Synergy HT, which we couldn't find from the setting panel.