Team:OUC-China/InterLab
Interlab
Background
Fluorescence data can not be directly compared, it is difficult to repeat the measurement. Different instruments, different settings and different measurement environments make this difficult. Through the Interlab study, we expect to have uniform fluorescence measurement steps and standards. These protocols solve this problem by establishing absolute units on the plate reader.
Design
OD 600 Reference point
Using LUDOX-S40 as a reference, the ratiometric conversion factor of Abs and OD600 can be obtained.
Protocol fluorescein fluorescence standard curve
By means of the gradient of the fluorescence standard curve, a method of obtaining a fluorescence correction of the cell can be obtained.
Cell measurement
At four time points, the fluorescence intensity of the eight circuits was measured.
Materials and Method
Plasmids used
Kit Plate 6 InterLab Part Locations
Positive Control (BBa_I20270): well 20B
Negative Control (BBa_R0040): well 20D
Test Device 1(BBa_J364000): well 20F
Test Device 2(BBa_J364001): well 20H
Test Device 3(BBa_J364002): well 20J
Test Device 4(BBa_J364003): well 20L
Test Device 5(BBa_J364004): well 20N
Test Device 6(BBa_J364005): well 20P
Strain
E.coli Competent DH5α （ TaKaRa Code : D9057）
Materials
1ml LUDOX (provided in kit) H20
Fluorescein (provided in kit) 10ml 1xPBS96 well plate, black with flat bottom
LB (Luria Bertani) media Chloramphenicol
50 ml Falcon tube Incubator at 37°C 1.5 ml eppendorf tubes for sample storage Ice bucket with ice Pipettes
Machines
Tecan Spark™ 10M
Software
Microsoft Office EXCEL
Protocol
InterLab_2017_Plate_Reader_ProtocolResults
OD 600 Reference point
| LUDOX-HS40 | H2O | |
|---|---|---|
| Replicate 1 | 0.0497 | 0.0422 |
| Replicate 2 | 0.0518 | 0.0495 |
| Replicate 3 | 0.0546 | 0.0428 |
| Replicate 4 | 0.0521 | 0.0569 |
| Arith. Mean | 0.05205 | 0.04785 |
| Corrected Abs600 | 0.0042 | |
| Reference OD600 | 0.0425 | |
| OD600/Abs600 | 10.1190476 |
Table 1. OD600 Reference Point
Protocol fluorescein fluorescence standard curve
Figure 1.Fluorescein fluorescence standard curve
Cell measurement
| 0h | 2h | 4h | 6h | |||||
|---|---|---|---|---|---|---|---|---|
| Abs600 | FI | Abs601 | FI | Abs602 | FI | Abs603 | FI | |
| Negative Control (Colony 1) | 0.0301 | 7 | 0.1156 | 8 | 0.5106 | 14 | 0.7453 | 17 |
| Negative Control (Colony 2) | 0.0408 | 5 | 0.1352 | 8 | 0.5184 | 17 | 0.7326 | 16 |
| Positive Control (Colony 1) | 0.0308 | 28 | 0.0805 | 65 | 0.4715 | 476 | 0.7293 | 722 |
| Positive Control (Colony 2) | 0.0437 | 31 | 0.1498 | 136 | 0.4956 | 428 | 0.7049 | 677 |
| Test Device 1: J23101.BCD2.E0040.B0015 (Colony 1) | 0.0283 | 46 | 0.0883 | 152 | 0.4358 | 606 | 0.6846 | 948 |
| Test Device 1: J23101.BCD2.E0040.B0015 (Colony 2) | 0.0261 | 38 | 0.0969 | 119 | 0.3719 | 464 | 0.5960 | 738 |
| Test Device 2: J23106.BCD2.E0040.B0015 (Colony 1) | 0.0378 | 44 | 0.1120 | 164 | 0.5090 | 718 | 0.7145 | 1258 |
| Test Device 2: J23106.BCD2.E0040.B0015 (Colony 2) | 0.0223 | 16 | 0.0788 | 115 | 0.4045 | 506 | 0.5945 | 963 |
| Test Device 3: J23117.BCD2.E0040.B0015 (Colony 1) | 0.0323 | 1 | 0.0945 | 10 | 0.4482 | 22 | 0.5704 | 28 |
| Test Device 3: J23117.BCD2.E0040.B0015 (Colony 2) | 0.0156 | 4 | 0.0537 | 8 | 0.3399 | 18 | 0.5254 | 24 |
| Test Device 4: J23101+I13504 (Colony 1) | 0.0201 | 9 | 0.0585 | 76 | 0.3224 | 339 | 0.4975 | 472 |
| Test Device 4: J23101+I13504 (Colony 2) | 0.0257 | 16 | 0.0628 | 84 | 0.3267 | 340 | 0.4900 | 471 |
| Test Device 5: J23106+I13504 (Colony 1) | 0.0263 | 7 | 0.0804 | 46 | 0.4000 | 106 | 0.5413 | 120 |
| Test Device 5: J23106+I13504 (Colony 2) | 0.0269 | 6 | 0.0850 | 45 | 0.3789 | 94 | 0.5285 | 111 |
| Test Device 6: J23117+I13504 (Colony 1) | 0.0265 | 1 | 0.0959 | 8 | 0.3967 | 15 | 0.4837 | 2 |
| Test Device 6: J23117+I13504 (Colony 2) | 0.0243 | -1 | 0.0883 | 5 | 0.4016 | 13 | 0.5212 | 10 |
Table 2. Raw data of Abs600 and Fluorescence measurement
| 0h | 2h | 4h | 6h | |||||
|---|---|---|---|---|---|---|---|---|
| average | SD | average | SD | average | SD | average | SD | |
| Negative Control (Colony 1) | 232 | 60 | 75 | 28 | 27 | 6 | 23 | 6 |
| Negative Control (Colony 2) | 114 | 11 | 59 | 25 | 34 | 4 | 22 | 3 |
| Positive Control (Colony 1) | 920 | 180 | 827 | 222 | 1013 | 75 | 994 | 87 |
| Positive Control (Colony 2) | 720 | 40 | 911 | 86 | 867 | 81 | 960 | 15 |
| Test Device 1: J23101.BCD2.E0040.B0015 (Colony 1) | 1636 | 324 | 1748 | 295 | 1394 | 80 | 1388 | 108 |
| Test Device 1: J23101.BCD2.E0040.B0015 (Colony 2) | 1462 | 64 | 1227 | 80 | 1248 | 64 | 1239 | 28 |
| Test Device 2: J23106.BCD2.E0040.B0015 (Colony 1) | 1190 | 212 | 1489 | 231 | 1413 | 69 | 1763 | 92 |
| Test Device 2: J23106.BCD2.E0040.B0015 (Colony 2) | 716 | 103 | 1469 | 163 | 1250 | 54 | 1621 | 83 |
| Test Device 3: J23117.BCD2.E0040.B0015 (Colony 1) | 27 | 61 | 112 | 50 | 49 | 7 | 49 | 11 |
| Test Device 3: J23117.BCD2.E0040.B0015 (Colony 2) | 282 | 108 | 148 | 45 | 52 | 3 | 45 | 11 |
| Test Device 4: J23101+I13504 (Colony 1) | 461 | 76 | 1319 | 170 | 1052 | 27 | 947 | 86 |
| Test Device 4: J23101+I13504 (Colony 2) | 617 | 136 | 1350 | 189 | 1042 | 73 | 962 | 51 |
| Test Device 5: J23106+I13504 (Colony 1) | 280 | 175 | 576 | 77 | 266 | 16 | 221 | 14 |
| Test Device 5: J23106+I13504 (Colony 2) | 215 | 93 | 530 | 41 | 249 | 20 | 211 | 12 |
| Test Device 6: J23117+I13504 (Colony 1) | 34 | 73 | 78 | 41 | 38 | 8 | 4 | 8 |
| Test Device 6: J23117+I13504 (Colony 2) | -55 | 102 | 60 | 23 | 33 | 3 | 20 | 4 |
Table 3. Data of Fluorescence/Abs600
Figure 2.Average level of devices
Discussion
Device 2 and device 3 have strong fluorescence. Device 4 and device 5 have moderate fluorescence intensity. The device 3 and the device 6 are weakly fluorescent.
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