Reproducibility and repeatability are key components to all engineering disciplines. The iGEM InterLab Study aims to develop a robust measurement procedure for green fluorescent protein (GFP) by having labs all over the world follow the same protocol and data analysis forms designed to yield absolute units of GFP measurement in a plate reader. One of the biggest challenges in synthetic biology is comparison of GFP data, and by conducting one of the biggest worldwide studies in synthetic biology, we seek the answer to the following question – How close can the numbers be when fluorescence is measured all around the world?
iGEM Peshawar is proud and excited to have been part of such an endeavor, that brings together labs from all over the world, working together to make biology easier to engineer. Although we do not have a fluorescence plate reader in our lab and couldn’t find one in Peshawar, a few friends in Islamabad and Karachi opened the doors to their labs for us. And while the plate reader in Islamabad was not up to specification, we travelled all the way across the country so that we could use the one at the University of Karachi and make our contribution to the study!
The InterLab plate reader protocol can be found here.
We used the Bio-Tek Instruments Synergy HTX Multi-Mode reader for our experiments, with Excitation and Emission filters – 485/20nm, 528/20nm with bottom optic measurement and gain setting of 60. Our experimental data can be found below:
OD600 Reference Point
Corrected value is particle-only contribution
Reference value is for 100uL of LUDOX-HS40 in a well of a standard 96-well flat-bottom plate
Corrected value = scaling factor * measured value
|
LUDOX-HS40 |
H2O |
| Replicate 1 |
0.056 |
0.0049 |
| Replicate 2 |
0.084 |
0.069 |
| Replicate 3 |
0.104 |
0.073 |
| Replicate 4 |
0.107 |
0.078 |
| Arith. Mean |
0.08775 |
0.06725 |
| Corrected Abs600 |
0.0205 |
|
| Reference OD600 |
0.0425 |
|
| OD600/Abs600 |
2.0731707 |
|
Plate Pattern
This table depicts how our samples were organized on our plate:
| |
|
|
|
|
|
|
|
|
| A1 |
A2 |
A3 |
A4 |
A5 |
A6 |
A7 |
A8 |
A9 |
| B1 |
B2 |
B3 |
B4 |
B5 |
B6 |
B7 |
B8 |
B9 |
| C1 |
C2 |
C3 |
C4 |
C5 |
C6 |
C7 |
C8 |
C9 |
| D1 |
D2 |
D3 |
D4 |
D5 |
D6 |
D7 |
D8 |
D9 |
| E1 |
E2 |
E3 |
E4 |
E5 |
E6 |
E7 |
E8 |
E9 |
| F1 |
F2 |
F3 |
F4 |
F5 |
F6 |
F7 |
F8 |
F9 |
| G1 |
G2 |
G3 |
G4 |
G5 |
G6 |
G7 |
G8 |
G9 |
| H1 |
H2 |
H3 |
H4 |
H5 |
H6 |
H7 |
H8 |
H9 |
Values measured are fluorescence from 100uL of X uM fluorescein solution:
| uM Fluorescein |
50.00 |
25 |
12.5 |
6.25 |
3.125 |
1.5625 |
0.78125 |
0.390625 |
0.1953125 |
0.09765625 |
0.048828125 |
0 |
| Replicate 1 |
4415971 |
2969835 |
1803277 |
987830 |
525106 |
270807 |
138240 |
72393 |
36616 |
19206 |
9325 |
578 |
| Replicate 2 |
4426468 |
2963649 |
1736159 |
953422 |
508391 |
256929 |
150357 |
50001 |
30669 |
15649 |
7313 |
564 |
| Replicate 3 |
4427698 |
2959896 |
1804084 |
879841 |
496013 |
245113 |
133114 |
55610 |
29763 |
18367 |
6368 |
561 |
| Replicate 4 |
4403182 |
2950232 |
1738049 |
959269 |
508211 |
243146 |
129941 |
69047 |
33589 |
13353 |
7722 |
565 |
| Arith. Mean |
4418329.75 |
2960903 |
1770392.25 |
945090.5 |
509430.25 |
253998.75 |
137913 |
61762.75 |
32659.25 |
16643.75 |
7682 |
567 |
| Arith. Std.Dev. |
11387.32121 |
8209.860535 |
38447.11531 |
46023.92807 |
11948.75651 |
12752.02427 |
8972.982596 |
10681.09871 |
3102.13178 |
2667.912964 |
1233.397206 |
7.527726527 |
uM Fluorescein/a.u.
| uM Fluorescein/a.u. |
50.00 |
25 |
12.5 |
6.25 |
3.125 |
1.5625 |
0.78125 |
0.390625 |
0.1953125 |
0.09765625 |
0.048828125 |
| Mean um Fluorescein/a.u. |
1.13165E-05 |
8.44337E-06 |
7.06058E-06 |
6.61312E-06 |
6.1343E-06 |
6.15161E-06 |
5.6648E-06 |
6.32461E-06 |
5.98031E-06 |
5.86744E-06 |
6.35617E-06 |
| Mean of med-high levels: |
|
6.8806E-06 |
|
|
|
|
|
|
|
|
|
Fluorescence Raw Readings:
These tables summarize our finalized sets of Absorbance reading data over an incubation period of 6 hours with samples measured every 2 hours for a total of 4 time points:
Hour 0
| Hour 0: |
Neg. Control |
Pos. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
LB + Chlor (blank) |
| C1, R1 |
6672 |
7338 |
8691 |
10504 |
6995 |
9038 |
7514 |
6939 |
6638 |
| C1, R2 |
6592 |
7477 |
8235 |
10088 |
6932 |
8934 |
7670 |
7081 |
6593 |
| C1, R3 |
6851 |
7545 |
8404 |
10404 |
6888 |
8936 |
7601 |
7056 |
6815 |
| C1, R4 |
6761 |
7472 |
8684 |
10566 |
6887 |
8966 |
7623 |
6876 |
6514 |
| C2, R1 |
6780 |
7679 |
8859 |
8867 |
6862 |
9269 |
7119 |
6870 |
6563 |
| C2, R2 |
6902 |
7523 |
8998 |
8539 |
6755 |
9183 |
7185 |
6878 |
6479 |
| C2, R3 |
6728 |
7673 |
9230 |
8525 |
6785 |
8920 |
6739 |
6858 |
6479 |
| C2, R4 |
6825 |
7708 |
9196 |
8894 |
6818 |
9269 |
7592 |
6951 |
6500 |
Hour 2
| Hour 2: |
Neg. Control |
Pos. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
LB + Chlor (blank) |
| C1, R1 |
6144 |
8714 |
11575 |
15887 |
6357 |
13507 |
7945 |
6350 |
5657 |
| C1, R2 |
6168 |
8653 |
11537 |
16005 |
6432 |
12991 |
8036 |
6161 |
5641 |
| C1, R3 |
6403 |
15902 |
11723 |
15775 |
6531 |
13419 |
7888 |
6511 |
5610 |
| C1, R4 |
6548 |
8656 |
11718 |
15934 |
6396 |
13192 |
7987 |
6377 |
5572 |
| C2, R1 |
6221 |
10313 |
14910 |
15867 |
12868 |
5854 |
13423 |
6236 |
5809 |
| C2, R2 |
6352 |
10622 |
14985 |
13219 |
13211 |
5902 |
13446 |
6134 |
5798 |
| C2, R3 |
6199 |
10587 |
16468 |
634 |
13120 |
5865 |
13550 |
6129 |
5746 |
| C2, R4 |
6387 |
10566 |
16160 |
629 |
13516 |
6004 |
13753 |
6078 |
5732 |
Hour 4
| Hour 4: |
Neg. Control |
Pos. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
LB + Chlor (blank) |
| C1, R1 |
7632 |
19951 |
26863 |
43220 |
8186 |
23519 |
12070 |
7074 |
5875 |
| C1, R2 |
7942 |
20358 |
27672 |
43174 |
8178 |
24376 |
12213 |
7123 |
5997 |
| C1, R3 |
7619 |
19773 |
27772 |
42805 |
8108 |
22183 |
12159 |
7164 |
5994 |
| C1, R4 |
7545 |
19838 |
27360 |
42756 |
7702 |
23235 |
11891 |
7052 |
5858 |
| C2, R1 |
7520 |
25434 |
58319 |
56593 |
7764 |
32871 |
10324 |
7073 |
5880 |
| C2, R2 |
7492 |
25324 |
59366 |
57119 |
7432 |
32376 |
10172 |
6992 |
5856 |
| C2, R3 |
7424 |
24794 |
59244 |
56511 |
7453 |
31342 |
10043 |
6932 |
5713 |
| C2, R4 |
7216 |
23947 |
67544 |
55408 |
7430 |
30710 |
9749 |
6692 |
5424 |
Hour 6
| Hour 6: |
Neg. Control |
Pos. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
LB + Chlor (blank) |
| C1, R1 |
779 |
842 |
982 |
1062 |
1050 |
821 |
869 |
1120 |
936 |
| C1, R2 |
5228 |
16920 |
26125 |
56643 |
7416 |
29661 |
11213 |
6457 |
5157 |
| C1, R3 |
4513 |
14856 |
23249 |
50604 |
6821 |
27780 |
10240 |
5936 |
4681 |
| C1, R4 |
4512 |
14199 |
21482 |
46489 |
6195 |
25254 |
9293 |
5433 |
4276 |
| C2, R1 |
3678 |
12246 |
18496 |
39416 |
5334 |
21755 |
8407 |
4930 |
3912 |
| C2, R2 |
3302 |
16093 |
44671 |
37900 |
4463 |
20200 |
6317 |
4330 |
3579 |
| C2, R3 |
2856 |
14166 |
38401 |
32452 |
3888 |
17281 |
5654 |
3743 |
3079 |
| C2, R4 |
2535 |
11083 |
31220 |
26662 |
3323 |
14011 |
4694 |
3084 |
2634 |
Abs600 Raw Readings:
These tables summarize our finalized sets of Absorbance reading data over an incubation period of 6 hours with samples measured every 2 hours for a total of 4 time points:
Hour 0
| Hour 0: |
Neg. Control |
Pos. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
LB + Chlor (blank) |
| C1, R1 |
0.224 |
0.276 |
0.187 |
0.326 |
0.237 |
0.233 |
0.237 |
0.674 |
-0.059 |
| C1, R2 |
0.215 |
0.278 |
0.176 |
0.315 |
0.115 |
0.246 |
0.206 |
0.652 |
-0.045 |
| C1, R3 |
0.224 |
0.277 |
0.172 |
0.314 |
0.158 |
0.243 |
0.174 |
0.649 |
-0.058 |
| C1, R4 |
0.228 |
0.269 |
0.141 |
0.299 |
0.097 |
0.226 |
0.147 |
0.559 |
-0.051 |
| C2, R1 |
0.326 |
0.231 |
0.184 |
0.166 |
0.297 |
0.223 |
0.280 |
0.354 |
-0.047 |
| C2, R2 |
0.306 |
0.247 |
0.220 |
0.176 |
0.275 |
0.226 |
0.246 |
0.322 |
-0.047 |
| C2, R3 |
0.305 |
0.249 |
0.172 |
0.176 |
0.328 |
0.224 |
0.244 |
0.320 |
-0.046 |
| C2, R4 |
0.29 |
0.238 |
0.141 |
0.158 |
0.216 |
0.226 |
0.196 |
0.336 |
-0.051 |
Hour 2
| Hour 2: |
Neg. Control |
Pos. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
LB + Chlor (blank) |
| C1, R1 |
0.261 |
0.34 |
0.226 |
0.284 |
0.202 |
0.215 |
0.428 |
0.215 |
-0.015 |
| C1, R2 |
0.285 |
0.292 |
0.259 |
0.305 |
0.216 |
0.291 |
0.612 |
0.313 |
0.017 |
| C1, R3 |
0.29 |
0.294 |
0.259 |
0.308 |
0.216 |
0.293 |
0.621 |
0.311 |
0.019 |
| C1, R4 |
0.3 |
0.29 |
0.27 |
0.21 |
0.268 |
0.278 |
0.451 |
0.388 |
-0.007 |
| C2, R1 |
0.31 |
0.277 |
0.239 |
0.278 |
0.231 |
0.35 |
0.26 |
0.316 |
-0.015 |
| C2, R2 |
0.278 |
0.264 |
0.221 |
0.259 |
0.224 |
0.336 |
0.246 |
0.293 |
-0.003 |
| C2, R3 |
0.281 |
0.267 |
0.223 |
0.257 |
0.227 |
0.337 |
0.246 |
0.295 |
-0.01 |
| C2, R4 |
0.3 |
0.29 |
0.226 |
0.27 |
0.219 |
0.33 |
0.27 |
0.312 |
-0.007 |
Hour 4
| Hour 4: |
Neg. Control |
Pos. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
LB + Chlor (blank) |
| C1, R1 |
0.293 |
0.297 |
0.374 |
0.325 |
0.286 |
0.315 |
0.282 |
0.349 |
-0.021 |
| C1, R2 |
0.29 |
0.289 |
0.363 |
0.316 |
0.279 |
0.304 |
0.267 |
0.334 |
-0.031 |
| C1, R3 |
0.291 |
0.29 |
0.363 |
0.317 |
0.279 |
0.305 |
0.266 |
0.335 |
-0.018 |
| C1, R4 |
0.287 |
0.301 |
0.382 |
0.32 |
0.292 |
0.317 |
0.291 |
0.332 |
-0.015 |
| C2, R1 |
0.239 |
0.407 |
0.594 |
0.363 |
0.404 |
0.344 |
0.316 |
0.247 |
-0.001 |
| C2, R2 |
0.226 |
0.394 |
0.566 |
0.345 |
0.389 |
0.332 |
0.296 |
0.223 |
0 |
| C2, R3 |
0.228 |
0.394 |
0.563 |
0.345 |
0.389 |
0.333 |
0.296 |
0.228 |
-0.002 |
| C2, R4 |
0.232 |
0.403 |
0.555 |
0.342 |
0.381 |
0.332 |
0.304 |
0.238 |
-0.002 |
Hour 6
| Hour 6: |
Neg. Control |
Pos. Control |
Device 1 |
Device 2 |
Device 3 |
Device 4 |
Device 5 |
Device 6 |
LB + Chlor (blank) |
| C1, R1 |
0.362 |
0.366 |
0.331 |
0.38 |
0.288 |
0.365 |
0.693 |
0.383 |
0.021 |
| C1, R2 |
0.357 |
0.364 |
0.331 |
0.377 |
0.288 |
0.363 |
0.684 |
0.389 |
0.019 |
| C1, R3 |
0.35 |
0.336 |
0.293 |
0.331 |
0.293 |
0.382 |
0.621 |
0.365 |
0.009 |
| C1, R4 |
0.36 |
0.385 |
0.347 |
0.39 |
0.29 |
0.385 |
0.629 |
0.399 |
0.013 |
| C2, R1 |
0.353 |
0.339 |
0.295 |
0.329 |
0.536 |
0.409 |
0.318 |
0.367 |
-0.013 |
| C2, R2 |
0.35 |
0.336 |
0.293 |
0.331 |
0.534 |
0.408 |
0.319 |
0.365 |
-0.014 |
| C2, R3 |
0.357 |
0.364 |
0.331 |
0.377 |
0.288 |
0.413 |
0.323 |
0.389 |
-0.021 |
| C2, R4 |
0.382 |
0.349 |
0.311 |
0.35 |
0.552 |
0.422 |
0.332 |
0.388 |
-0.023 |
