Team:Aix-Marseille/Measurement

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Measurement

To quantify the fluorescent molecules in the bacteria, we followed the standard iGEM protocols for RFP and GFP (1).

To use this, we have chosen to modify the protocols to have reproducible and standardized results. In measuring, the quality of experiments reproducibility is a key. The reproducibility of data is a measure of whether results in a paper can be attained by a different research team, using the same methods. This shows that the results obtained are not artifacts of the unique setup in one research lab. We wanted to obtain results over a wide range of concentrations.

Here we present what we think can be rewarded as a Special Prize for ARBO-BLOCK project. The improvement of the Protocol—Plate Reader Fluorescence Calibration Protocol will allow future iGEM teams to use it and generate reproducible and standardized results.

We had to choose a gain for an expected signal -the gain determines the amplification of a detected signal- and we had to create programs with a range of gains to make sure we were in the detection range of the device.

Gain is a way to control the voltage across a microplate, making the device more or less sensitive to the intensity of the signal being measured. As fluorescence signal intensities can vary considerably, gain provides greater flexibility and allows a microplate reader to measure a wider dynamic range of analyte concentrations and test responses.

For the choice of the right gain, we had to consider two assumptions:

  • High gain for better sensitivity, when the signal is low, but we still want to observe a signal
  • Lower gain to be able to observe much higher concentration values.


For the choice of the gain, we made several tests on fluorescein. Indeed, we need the fluorescence signal for our manipulations, but we also chose this because it is a product that all teams can get. Our fluorescein was at a concentration of 10-3 mol.L-1.

We made different dilutions on a logarithmic scale; we used the following concentrations:

Table 1: Concentrations of fluorescein used (mol.L-1). Concentration used (mol.L-1)
10-7 5.10-8 2.10-8 10-8 5.10-9 2.10-9 10-9 5.10-10
2.10-10 10-10 5.10-11 2.10-11 10-11 5.10-12 2.10-12 10-12

In addition, we studied a range of gains: 1, 7, 25, 49, 50, 63 and 70 .

To read the gain measurement, we always used the same microplate, the plan is shown below.

Table 2: Microplate plan. The samples are placed in rows (A to G) and columns (1 to 12). Are presented: the different concentrations of fluorescein used (mol.L-1), in italic the "blank" (negative control: LB) and underligned the "empty" (negative control: empty well). The analyses were done in duplicate or triplicate .
1 2 3 4 5 6 7 8 9 10 11 12
A LB LB 10-7 10-7 5.10-8 5.10-8 5.10-8 2.10-8 2.10-8 10-8 10-8 5.10-9
B 5.10-9 5.10-9 2.10-9 2.10-9 10-9 10-9 5.10-10 5.10-10 5.10-10 2.10-10 2.10-10 10-10
C 10-10 5.10-11 5.10-11 5.10-11 2.10-11 2.10-11 10-11 10-11 5.10-12 5.10-12 5.10-12 2.10-12
D 2.10-12 10-12 10-12 10-12 LB LB 10-7 10-7 5.10-8 5.10-8 5.10-8 2.10-8
E 2.10-8 10-8 10-8 5.10-9 5.10-9 5.10-9 2.10-9 2.10-9 10-9 10-9 5.10-10 5.10-10
F 5.10-10 2.10-10 2.10-10 10-10 10-10 5.10-11 5.10-11 5.10-11 2.10-11 2.10-11 10-11 10-11
G 5.10-12 5.10-12 5.10-12 2.10-12 2.10-12 10-12 10-12 10-12 LB LB Empty Empty

We obtained the following results: these results allow us to place this in the best range of gain to have the most reliable results.

Table 3: Summary table of observed fluorescein gain measurements.
Gains Minimum observed measure Maximum observed measure
1 / /
7 / /
35 / 500
49 20 5000
50 13 6000
56 60 13000
63 100 33000
70 300 55000

When we were using the microplate reader, we realised that some gains were too weak, and we had results under 0 (/).

For this reason, we chose to study fluorescein at gain 35, 50 and 70. However, the 70 gain was not necessary in our study as the fluorescein levels increased too fast and it was beyond the capabilities of the device.

Our study therefore stopped at gains 35 and 50 because these were the best gains for the concentrations studied.

The results for the TECAN device using our program at gain 35 are presented in the Table 4.

Table 4: Microplate gain 35 measurement results of fluorescein.The samples are placed in rows (A to G) and columns (1 to 12). Are presented: the gain measurement obtained at different concentrations of fluorescein used, in italic the "blank" (negative control: LB) and underligned "empty" (negative control: empty well). The analyses were done in duplicate or triplicate.
1 2 3 4 5 6 7 8 9 10 11 12
A -3 -3 468 440 355 369 326 126 113 69 67 65
B 34 26 12 11 4 5 1 1 2 -1 -2 -1
C -2 -2 -2 8 1 4 -1 -1 -2 -2 -2 -2
D -3 -3 -3 -3 -3 -3 476 430 266 247 244 78
E 97 63 71 40 39 38 13 12 5 5 0 1
F 1 -1 -1 -2 -2 -1 -1 -2 -2 -1 -2 -3
G -3 -3 -3 -3 -2 -4 -3 -3 -3 -3 -4 -4

Figure 1: Fluorescein concentration (nM) according to the fluorescence signal (gain 35, mAU).

For the graph of fluorescein concentration according to the fluorescence signal at gain 35, we used a concentration in nmole/L and we added the error bars (standard deviation).

We can therefore observe that the results form a linear line with the following equation:
y= 4,7622 x +7,049

The results for the TECAN device using our program at gain 50 are presented in the table 5.

Table 5: Microplate gain 50 measurement results of fluorescein . The samples are placed in rows (A to G) and columns (1 to 12). Are presented: the gain measurement obtained at different concentrations of fluorescein used, in italic the "blank" (negative control: LB) and in underlined "empty" (negative control: empty well). The analyses were done in duplicate or triplicate.
1 2 3 4 5 6 7 8 9 10 11 12
A 23 24 5953 5765 4449 4561 4020 1617 1461 908 880 663
B 491 390 204 192 108 125 70 67 75 47 45 50
C 39 36 39 172 82 105 49 50 41 30 31 30
D 27 26 28 30 28 29 6127 5617 3468 3180 3137 1077
E 1285 848 945 551 528 528 225 200 118 116 66 72
F 75 56 53 38 38 44 44 38 37 34 32 33
G 29 38 34 27 28 26 26 26 24 24 13 14

Figure 2: Fluorescein concentration (nM) according to the fluorescence signal (gain 50, mAU).

For the graph of fluorescein concentration according to the fluorescence signal at gain 50, we used a concentration in nmole./L and we added the error bars (standard deviation).

We can therefore observe that the results form a linear line with the following equation:
y= 60,786 x + 101,65

In conclusion, our protocols are therefore reproducible and standardised.

The modifications made and the study of the different gains allowed us to obtain reliable results over a wider dynamic range. All our final fluorescence measurements are therefore reproducible and standardized to the equivalent number of molecules per cell.

Knowing the linear equation allows to relate a physical measurement (intensity of fluorescence according to the gain) to a biological value (the concentration produced in the sample).

Thus, obtained results of the standardization curve permit to know an unknown concentration thanks to a fluorescence value.

References

  1. Baldwin, G., Haddock-Angelli, T., Beal, J., Dwijayanti, A., Storch, M., Farny, N., Telmer, C., Vignoni, A., Tennant, R., & Rutten, P. (2019, September 5). Protocol—Plate Reader Fluorescence Calibration. Protocols.Io.

IGEM Aix-Marseille 2021