Team:UiOslo Norway/InterLab

The Interlab study

Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?


A big part of synthetic biology is to detect and eliminate the huge differences one can get while doing the same types of experiments across different labs. Without being able to recreate and compare results as if they all where performed in the same lab, one still can not completely rely on others results, which makes further researching and comparing of data difficult. A big part of the iGEM competition over the last years was the Interlab study. This project is created in a way that different teams across the world can recreate the same lab experiments and then be able to compare the similarities or differences that might arise during the procedure. This year’s Interlab is focusing on the reliability of cell counting methods to detect the variability that may occur during fluorescence measurements.

A plate reader is an instrument that allows us to detect biological matter by measuring light that is emitted by the substrate (1). For the purpose of the study we want to be able to measure the cell density by using bacteria that has the ability to fluoresce and compare the results by plating them out and counting the amount of CFU (colony forming units) instead of relying on OD. Each team is distributed a plate kit with eight different plasmids. Each of them contain differing ability to express GFP. These plasmids were each transformed into DH5-alpha Escherichia coli cells, which were then able to express GFP. When the protein GFP is exposed to ultraviolet light it emits fluorescence that can be measured by light detecting instruments like a plate reader (2).


The eight plasmids distributed were:

Part numberDeviceAnderson Promoter strength (n/a) (3)
BBa_R0040Negative control-
BBa_I20270Positive control-
BBa_J364000Test device 10.70
BBa_J364001Test device 20.47
BBa_J364002Test device 30.06
BBa_J364007Test device 41.00
BBa_J364008Test device 50.72
BBa_J364009Test device 60.16

The differences in the promoter strengths will be reflected in the measurement in the plate reader, but this has been a more important comparison in earlier Interlab studies.

The individual devices where all transformed into separate DH5-alpha cells and incubated on petri dishes with LB agar and chloramphenicol overnight. From each plate we made an overnight culture of 2 colonies (16 overnight cultures in total). We diluted these cultures to an Abs600=0.02. 500 µl was taken out of the cultures and stored on ice. These samples were used for t=0 h. The rest of the culture was incubated at 37o C for 6 hours. This culture marked the 6 hour ending point. Fluorescence was then measured for both t=0 h and t=6 h time points.

We used three replicates of the positive and negative devices at t=0 h for a further dilution of an OD600=0.1. These triplicates were used for a dilution series where the last three dilutions where plated (dilution with dilution factors 8x104, 8x105 and 8x106 where used). The colonies on these plates were counted and CFU/ml at an OD600=0.1 where calculated by taking into count the dilution factor.

Organization is key to a successful experiment


Particle standard curve

A particle standard curve was made with silica beads of known size and concentration. The curve should in theory correlate to the amount of cells in the solution measured, so that a measured absorption correspond to a calculated amount of cells in the solution.

Particle standard curve
Negative controls
SampleDilution factorColoniesCFU / ml
1.18 x 104>300-
1.18 x 1052471.97 x 108
1.18 x 106342.72 x 108
1.28 x 104>300-
1.28 x 1051371.09 x 108
1.28 x 106443.52 x 108
1.38 x 104>300-
1.38 x 1052231.78 x 108
1.38 x 106473.76 x 108
2.18 x 104>300-
2.18 x 1053482.78 x 108
2.18 x 106685.44 x 108
2.28 x 104>300-
2.28 x 1051911.53 x 108
2.28 x 106604.80 x 108
2.38 x 104>300-
2.38 x 1052672.14 x 108
2.38 x 106151.20 x 108
Positive controls
SampleDilution factorColoniesCFU / ml
1.18 x 104>300-
1.18 x 1051431.14 x 108
1.18 x 10664.80 x 108
1.28 x 104>300-
1.28 x 105887.04 x 108
1.28 x 106252.00 x 108
1.38 x 104>300-
1.38 x 105826.56 x 107
1.38 x 106272.16 x 108
2.18 x 104>300-
2.18 x 1051219.68 x 107
2.18 x 106231.84 x 108
2.28 x 104>300-
2.28 x 1051731.38 x 108
2.28 x 106118.80 x 107
2.38 x 104>300-
2.38 x 1051471.17 x 108
2.38 x 106131.04 x 108

Fluorescein standard curve

Rep. 14.81x1043.79x1042.81x1041.70x1049.60x1035.14x1032.61x1031.29x1036.57x1023.23x1021.70x1020
Rep. 24.99x1043.99x1042.78x1041.69x1049.12x1034.81x1032.53x1031.34x1036.61x1023.35x1021.72x1020
Rep. 34.92x1043.94x1042.80x1041.73x1049.50x1035.25x1032.60x1031.20x1036.27x1023.09x1021.59x1020
Rep. 44.95x1044.03x1042.85x1041.70x1049.54x1035.06x1032.56x1031.32x1036.64x1023.20x1021.75x1020
Arith. Mean4.92x1043.94x1042.81x1041.71x1049.44x1035.06x1032.57x1031.29x1036.52x1023.22x1021.69x1020
Arith. Std.Dev.7.78x1021.05x1032.64x1021.39x1022.15x1021.87x1023.69x1016.29x1011.71x1011.07x1016.98x1000
Arith. Net Mean4.92x1043.94x1042.81x1041.71x1049.44x1035.06x1032.57x1031.29x1036.52x1023.22x1021.69x102

Abs600 Raw Readings

Abs600 measured of all eight constructs in a replicates of eight at both time 0h and 6h (Raw readings)

0 hours         
SampleNeg. ControlPos. ControlDevice 1Device 2Device 3Device 4Device 5Device 6LB + Chlor (blank)
6 hours         
SampleNeg. ControlPos. ControlDevice 1Device 2Device 3Device 4Device 5Device 6LB + Chlor (blank)



Feedback for the InterLab study

We enjoyed participating in the InterLab Study, but it was not without problems. Here we share some of our thoughts regarding this years study

Some parts of the general protocol was unclear. A clear, concise list of things to do and materials required would be much appreciated.

Compulsory forms and protocols were not available early on, when there was no other labwork to be done. Late publications may cause the InterLab Study to crash with work on our project.

After submitting the online forms, we were not able to review them after submission. Even though we could not change the content, it would be nice to look back on what we submitted in terms of documentation.