Difference between revisions of "Team:ZJUT-China/InterLab"

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Revision as of 09:42, 27 July 2018

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JUDGING FORM ⇗

INTERLAB

Our team participated in the Fifth International InterLaboratory Measurement Study this year. The interlab study aims to solve the problem of reliability and repeatability in synthetic biology study. This year we were asked to measure green fluorescent protein, which is used as a measurement marker. We followed the experiment protocol strictly to make sure our data is valid. After two weeks lab work, we got the data we need after several attempts.

Calibration Protocol

Three calibration measurement were done by the calibration protocol, including an OD reference point at 600nm, one particle standard curve and one fluorescent standard curve.

(Plate Reader: Molecular Device, Spectramax M5)

★ OD600 reference point ★


LUDOX CL-X H2O
Replicate 1 0.066 0.0285
Replicate 2 0.059 0.029
Replicate 3 0.068 0.026
Replicate 4 0.064 0.028
Arith. Mean 0.064 0.028
Corrected Abs600 0.036
Reference OD600 OD600
OD600/Abs600 1.728


Particle_Standard_Curve.png

Form1.Particle standard curve

Particle_Standard_Curve_(log_scale)e

Form2.Fluorescein standard curve



Fluorescein_Standard_Curve Fluorescein_Standard_Curve_%28log_scale%29

Cell measurement

Transformation

Transform Escherichia coli DH5α with these following plasmids:

Device Part Number Location
Positive control BBa_R0040 Well 2D
Negative control Ba_I20270 Well 2B
Test Device 1 BBa_J364000 Well 2F
Test Device 2 BBa_J364001 Well 2H
Test Device 3 BBa_J364002 Well 2J
Test Device 4 BBa_J364007 Well 2L
Test Device 5 BBa_J364008 Well 2N
Test Device 6 BBa_J364009 Well 2P

The transformation used E.coli DH5α competent cells bought from Tsingke Biological Technology company, and follow the steps in http://parts.igem.org/Help:2017_DNA_Distribution to use the DNA in the Distribution Kit.

Measurement


For this part of inter lab study, we followed the following protocols:

1.Grown 8 devices in incubator for 12 hrs at 37 ℃
2.Pick 2 colonies from each of plate and inocubate them on 10mL LB medium with Chloramphenicol. Grow the cells for 16-20hrs at 37°C and 180rpm.
3.Measure OD600nm of the overnight cultures and record the data, then dilute to target OD600nm = 0.02 in the falcon tubes.
4.Measure the OD600nm and Fl under the same condition as standard curve measurement and use the same 96 wells plates.


plate_lay_out

★ 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 98.891 514.509 170.45 453.211 107.527 377.998 110.335 121.276 100.56
Colony 1, Replicate 2 87.951 520.569 155.638 443.722 103.159 353.38 99.569 117.851 97.983
Colony 1, Replicate 3 89.665 500.61 151.21 474.651 99.334 358.843 97.329 115.674 97.686
Colony 1, Replicate 4 88.651 553.489 158.106 471.047 107.222 391.116 102.558 132.724 104.901
Colony 2, Replicate 1 94.201 799.161 176.469 396.183 105.65 452.456 105.862 129.379 100.254
Colony 2, Replicate 2 97.455 750.414 168.71 425.555 106.286 462.598 109.024 125.311 101.914
Colony 2, Replicate 3 95.836 795.354 174.674 405.952 103.704 444.673 106.365 128.606 105.761
Colony 2, Replicate 4 100.733 804.913 203.631 370.775 117.227 506.476 113.511 125.021 105.18
Hour 6: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6 LB + Chlor (blank)
Colony 1, Replicate 1 142.426 1579.487 407.651 2268.46 167.703 1014.837 144.06 365.966 130.779
Colony 1, Replicate 2 143.238 1593.226 396.943 2328.22 174.862 1003.434 141.231 339.499 126.638
Colony 1, Replicate 3 151.515 1699.317 396.669 2175.501 169.71 977.6 143.762 347.711 127.911
Colony 1, Replicate 4 155.799 1783.066 406.205 2240.128 171.295 1044.087 154.138 340.887 127.237
Colony 2, Replicate 1 150.46 2062.959 354.432 2090.23 165.189 1268.176 150.674 320.936 120.878
Colony 2, Replicate 2 145.579 2023.17 366.367 2169.758 167.611 1203.787 144.458 322.957 127.103
Colony 2, Replicate 3 146.553 2101.747 353.019 1926.808 183.724 1172.553 158.135 332.951 131.201
Colony 2, Replicate 4 157.503 2071.459 316.402 1750.215 179.275 1253.449 155.625 327.385 137.828


★ 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 0.1306 0.0884 0.0767 0.1068 0.1181 0.0753 0.1067 0.0866 0.0486
Colony 1, Replicate 2 0.1272 0.0906 0.0737 0.1075 0.1184 0.0754 0.108 0.0896 0.0497
Colony 1, Replicate 3 0.1246 0.09 0.0747 0.1119 0.1214 0.0731 0.1083 0.0879 0.0464
Colony 1, Replicate 4 0.1225 0.0872 0.0743 0.1109 0.1247 0.0746 0.1079 0.0903 0.0476
Colony 2, Replicate 1 0.1301 0.0907 0.0701 0.1109 0.1132 0.0813 0.1103 0.0909 0.0468
Colony 2, Replicate 2 0.1327 0.0919 0.0685 0.1125 0.1145 0.0822 0.1081 0.0896 0.0472
Colony 2, Replicate 3 0.1294 0.0952 0.0693 0.1151 0.1139 0.0809 0.1084 0.0898 0.05
Colony 2, Replicate 4 0.1202 0.0941 0.0692 0.1105 0.1134 0.0805 0.1092 0.089 0.0472
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 0.9386 0.7564 0.1978 0.911 0.7963 0.2566 0.8131 0.6363 0.0444
Colony 1, Replicate 2 0.9413 0.7528 0.1974 0.9012 0.8211 0.2547 0.7951 0.5996 0.0438
Colony 1, Replicate 3 0.9745 0.7748 0.1951 0.8642 0.81 0.2536 0.7994 0.6044 0.0458
Colony 1, Replicate 4 0.9932 0.7975 0.2009 0.8969 0.8021 0.2622 0.8266 0.6092 0.0438
Colony 2, Replicate 1 0.8246 0.78 0.1031 0.8625 0.8193 0.2834 0.7633 0.5251 0.0457
Colony 2, Replicate 2 0.8327 0.7665 0.1031 0.9018 0.8211 0.2781 0.7394 0.5374 0.0462
Colony 2, Replicate 3 0.8062 0.7927 0.1005 0.829 0.8082 0.276 0.7689 0.5668 0.0458
Colony 2, Replicate 4 0.8262 0.7805 0.1005 0.8787 0.8156 0.2928 0.7604 0.5332 0.0477


★ uM Fluorescein / OD ★

Hour 0: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6
olony 1, Replicate 1 -0.002 0.872 0.208 0.508 0.008 0.871 0.014 0.046
Colony 1, Replicate 2 -0.011 0.866 0.201 0.501 0.006 0.833 0.002 0.042
Colony 1, Replicate 3 -0.009 0.775 0.159 0.482 0.002 0.820 0.000 0.036
Colony 1, Replicate 4 -0.018 0.950 0.167 0.485 0.003 0.889 -0.003 0.055
Colony 2, Replicate 1 -0.006 1.334 0.274 0.387 0.007 0.856 0.007 0.055
Colony 2, Replicate 2 -0.004 1.216 0.263 0.415 0.005 0.864 0.010 0.046
Colony 2, Replicate 3 -0.010 1.279 0.299 0.387 -0.003 0.919 0.001 0.048
Colony 2, Replicate 4 -0.005 1.251 0.375 0.352 0.015 1.010 0.011 0.040
Hour 6: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6
Colony 1, Replicate 1 0.001 0.171 0.151 0.207 0.004 0.349 0.001 0.033
Colony 1, Replicate 2 0.002 0.173 0.148 0.215 0.005 0.348 0.002 0.032
Colony 1, Replicate 3 0.002 0.181 0.151 0.210 0.005 0.343 0.002 0.033
Colony 1, Replicate 4 0.003 0.184 0.149 0.208 0.005 0.352 0.003 0.032
Colony 2, Replicate 1 0.003 0.222 0.341 0.202 0.005 0.405 0.003 0.035
Colony 2, Replicate 2 0.002 0.221 0.352 0.200 0.004 0.389 0.002 0.033
Colony 2, Replicate 3 0.002 0.221 0.340 0.192 0.006 0.379 0.003 0.032
Colony 2, Replicate 4 0.002 0.221 0.283 0.163 0.005 0.382 0.002 0.033


★ Net Fluorescein a.u. ★

Hour 0: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6
Colony 1, Replicate 1 -1.67 413.95 69.89 352.65 6.97 277.44 9.77 20.72
Colony 1, Replicate 2 -10.03 422.59 57.66 345.74 5.18 255.40 1.59 19.87
Colony 1, Replicate 3 -8.02 402.92 53.52 376.97 1.65 261.16 -0.36 17.99
Colony 1, Replicate 4 -16.25 448.59 53.21 366.15 2.32 286.22 -2.34 27.82
Colony 2, Replicate 1 -6.05 698.91 76.22 295.93 5.40 352.20 5.61 29.13
Colony 2, Replicate 2 -4.46 648.50 66.80 323.64 4.37 360.68 7.11 23.40
Colony 2, Replicate 3 -9.93 689.59 68.91 300.19 -2.06 338.91 0.60 22.85
Colony 2, Replicate 4 -4.45 699.73 98.45 265.60 12.05 401.30 8.33 19.84
Hour 6: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6
Colony 1, Replicate 1 11.65 1448.71 276.87 2137.68 36.92 884.06 13.28 235.19
Colony 1, Replicate 2 16.60 1466.59 270.31 2201.58 48.22 876.80 14.59 212.86
Colony 1, Replicate 3 23.60 1571.41 268.76 2047.59 41.80 849.69 15.85 219.80
Colony 1, Replicate 4 28.56 1655.83 278.97 2112.89 44.06 916.85 26.90 213.65
Colony 2, Replicate 1 29.58 1942.08 233.55 1969.35 44.31 1147.30 29.80 200.06
Colony 2, Replicate 2 18.48 1896.07 239.26 2042.66 40.51 1076.68 17.36 195.85
Colony 2, Replicate 3 15.35 1970.55 221.82 1795.61 52.52 1041.35 26.93 201.75
Colony 2, Replicate 4 19.68 1933.63 178.57 1612.39 41.45 1115.62 17.80 189.56


★ Net Abs 600 ★

Hour 0: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6
Colony 1, Replicate 1 0.082 0.040 0.028 0.058 0.070 0.027 0.058 0.038
Colony 1, Replicate 2 0.078 0.041 0.024 0.058 0.069 0.026 0.058 0.040
Colony 1, Replicate 3 0.078 0.044 0.028 0.066 0.075 0.027 0.062 0.042
Colony 1, Replicate 4 0.075 0.040 0.027 0.063 0.077 0.027 0.060 0.043
Colony 2, Replicate 1 0.083 0.044 0.023 0.064 0.066 0.035 0.064 0.044
Colony 2, Replicate 2 0.086 0.045 0.021 0.065 0.067 0.035 0.061 0.042
Colony 2, Replicate 3 0.079 0.045 0.019 0.065 0.064 0.031 0.058 0.040
Colony 2, Replicate 4 0.073 0.047 0.022 0.063 0.066 0.033 0.062 0.042
Hour 6: Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6
Colony 1, Replicate 1 0.894 0.712 0.153 0.867 0.752 0.212 0.769 0.592
Colony 1, Replicate 2 0.898 0.709 0.154 0.8576 0.777 0.211 0.751 0.55
Colony 1, Replicate 3 0.929 0.729 0.149 0.818 0.764 0.208 0.754 0.559
Colony 1, Replicate 4 0.949 0.754 0.157 0.853 0.758 0.218 0.783 0.565
Colony 2, Replicate 1 0.779 0.734 0.057 0.817 0.774 0.238 0.718 0.479
Colony 2, Replicate 2 0.787 0.720 0.057 0.856 0.775 0.232 0.693 0.491
Colony 2, Replicate 3 0.760 0.747 0.055 0.783 0.762 0.230 0.723 0.521
Colony 2, Replicate 4 0.779 0.733 0.053 0.831 0.768 0.245 0.713 0.486

CFU Protocol

For this part of inter lab study, we followed the following protocol:


1.Pick two colonies from positive control device and negative control device, incubate overnight.
2.Prepare Starting Samples: Dilute the overnight cultures 1:8, measurement then dilute further to OD600nm = 0.1
Calculation:
Use (C1)(V1) = (C2)(V2) to calculate your dilutions C1 is your starting OD600   C2 is your target OD600 of 0.1
V1 is the unknown volume in μL   V2 is the final volume of 1000 μL
3.Check and make sure OD600 = 0.1
4.Dilute Dilute
5.Incubate at 37 degree Celsius overnight for 18-20 hrs. Then count colony number.
colony_number