Team:UNOTT/InterLab
INTERLAB STUDY:
Introduction
Our team decided to take part in the iGEM interlab study this year. This study
is organised by iGEM and is aimed at tackling a prominent issue regarding experimental fluorescent reporter data. At present, experiments using fluorescent reporters are almost incomparable; separate groups often interpret fluorescence measurements in largely different ways, making it hard to properly compare results. To combat this, the interlab study is
performed each year, iteratively refining an optimal protocol for fluorescence measurement.
Theoretically, this would be followed by anyone and yield comparably robust units of fluorescence, facilitating more meaningful comparisons of data between researchers.
The study required us to measure a number of different fluorescent test devices sent to us by iGEM HQ, we followed their protocol and then shared the results we obtain. Teams around the world do the same; collectively, we provide a large set of data illustrating how similar the results were, potentially highlighting any aspects in which the protocol could be improved and, hopefully, showing that this is possible. This is quite an important issue, we felt we should participate and help in any way we could!
Methods
We were given 8 specimens to measure, termed “test devices”. These included a positive (BBa_I20270) and negative(BBa_R0040) control, and 6 different test devices (BBa_J36400, BBa_J364001, BBa_J364002, BBa_J364003, BBa_J364004, BBa_J364005).
First we transformed these into DH5α-E. coli cells, then selected two resulting colonies to be prepared for measurement. The two colonies were grown overnight.
But before the experiment could be undertaken, we needed to calibrate our equipment by performing standard reference measurements, enabling the conversion of our data to absolute values. Firstly, we measured a solution of half LUDOX-S40, half water; fluorescein was then serially diluted by half 10 times and measured to give a standard curve.
After 18 hours of growth, the test device sample's optical density was measured, a new batch of LB/Chloramphenicol was inoculated to produce an OD of 0.02. These were then grown at 37°C, OD600nm and fluorescence were measured at 0, 2, 4, and 6 hours.
For a full description of the methods, please visit the official interlab study page
Results
LUDOX-S40 and Fluoroscein Reference Measurements
LUDOX-HS40
H20
Replicate 1
S0.041
0.029
Replicate 2
0.045
0.03
Replicate 3
0.043
0.032
Replicate 4
0.041
0.032
Arith. Mean
0.0425
0.03075
Corrected Abs600
0.01175
Reference OD600
0.0425
OD600/Abs600
3.617
uM Fluoroscein/a.u
50.00 25 12.5 6.25 3.13 1.56 0.78 0.39 0.2 0.1 0.05 0
Replicate 1
26172 17413 1031 5606 2988 1534 795 407 223 124 77 29
Replicate 2
26021 17369 10299 5641 2997 1529 788 410 224 124 78 30
Replicate 3
26141 17333 10321 5602 2979 1527 787 409 225 126 78 30
Replicate 4
26243 17204 10273 5570 2966 1523 789 417 216 126 76 28
Arith. Mean
26144 17330 10306 5605 2983 1528 7890 411 222 125 77 29
Arith. Std. Dev.
92.59 89.99 25.74 29.044 13.22 4.57 3.59 4.34 4.08 1.15 0.96 0.96
uM Fluoroscein/a.u
50.00 25 12.5 6.25 3.13 1.56 0.78 0.39 0.2 0.1 0.05
Mean uM Fluorescein/a.u.(×10^-3)
1.9 1.4 1.2 1.1 1 1 0.99 0.95 0.88 0.78 0.63
Mean of med-high levels
0.001168
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 |
| Serial dilution of fluorescein intesnity plotted on linear scale | Fluorescein dilution of fluorescein intensity plotted on log (scale Graph) |
OD600nm and Fluorescence Results
| Fluorescence Data (colonies averaged and minus background) | Timepoints (Hours) | |||
|---|---|---|---|---|
| Test Device | 0H | 2H | 4H | 6H |
| Negative Control | 24.25 | 21 | 19.38 | 18.75 |
| Positive | 27.8 | 38.6 | 81.9 | 107.5 |
| 1 | 44.25 | 62.4 | 103.5 | 174.8 |
| 2 | 28 | 40.1 | 93.5 | 137.4 |
| 3 | 24 | 21.4 | 20.5 | 21.6 |
| 4 | 28 | 36.5 | 79.6 | 124 |
| 5 | 25 | 28.4 | 40.5 | 45.1 |
| 6 | 23.1 | 21 | 9.75 | 19.4 |
| OD600nm Data (colonies averaged and minus background) | ||||
|---|---|---|---|---|
| Test Device | 0H | 2H | 4H | 6H |
| Negative Control | 0.01 | 0.07 | 0.3 | 0.55 |
| Positive control | 0.01 | 0.06 | 0.26 | 0.55 |
| 1 | 0.01 | 0.02 | 0.03 | 0.07 |
| 2 | 0.001 | 0.05 | 0.22 | 0.5 |
| 3 | 0.07 | 0.06 | 0.25 | 0.48 |
| 4 | 0.01 | 0.03 | 0.13 | 0.4 |
| 5 | 0.08 | 0.05 | 0.23 | 0.54 |
| 6 | 0.08 | 0.61 | 0.25 | 0.5 |
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 |
| OD600nm measurements of all test devices (minus background), biological replicates were averaged. | Fluoresence measurements of all test devices (minus background), biological replicates were averaged. |
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| Graph depicting each test device's averaged fluorescence intensity minus background |
 |
| Graph depicting each test device's averaged OD600nm minus background |
