Team:Uppsala/Design
Design
In this iGEM project we faced the need to characterize a promoter induced by the NahR protein [1], which is a regulated promoter, induced by salicylate. We decided to compare it with different amounts of salicylate concentrations and also with two constitutive promoters (the J23101 and J23110). Among the existing approaches for measuring the strength of a promoter, we chose to use fluorometry. Even though we did not have access to a fluorometer in our lab, we decided to go through with this approach while searching for a solution. One of the solutions was to build our own fluorescence measurement instrument. Hence, some of our team members decided to work around the idea of constructing a custom fluorescence measurement instrument (or fluorometer), while others searching/asking for an available fluorometer around.
Fluorometry
Fluorometry is an analytical technique for identification and measurement of fluorescent compounds and targets like cells, proteins, nucleotides, or labeled targets with fluorescent tags [2]. It works by emitting light in a specific wavelength that excites electrons in the molecules of a respective compound. That causes the compound to emit light, typically within the visible spectrum. Instruments take advantage of this emitted light in order to measure the fluorescence level of the compound.
Fluorometry is an analytical technique for identification and measurement of fluorescent compounds and targets like cells, proteins, nucleotides, or labeled targets with fluorescent tags [2]. It works by emitting light in a specific wavelength that excites electrons in the molecules of a respective compound. That causes the compound to emit light, typically within the visible spectrum. Instruments take advantage of this emitted light in order to measure the fluorescence level of the compound.
Custom Fluorometer
Since there are a lot of instruments that can measure fluorescence, we had to choose one that suits our case. Depended on our low budget, we had to choose the best combination of a cheaper and easier approach, which as we concluded is the filter fluorometer. This type of fluorometer uses optical filters in order to let only the desirable wavelengths to pass, while blocking the rest of the spectrum [3].
Originally, filter fluorometers have one excitation filter between the light source and the sample, in order to let pass only the wavelength that excites the compound. Also, they have an emission filter in order to let only the wavelength emitted by the compound to pass out to a light sensor. The actual measurement is performed by the light sensor that captures the light’s intensity, that comes out of the emission filter. Based on that and taking advantage of existed materials, we had as a base of our fluorometer project the Arduino microcontroller [4]. The rest of the main fluorometer components are two green LEDs used as light source, one longpass optical filter at 590nm (FGL590) used as an emission filter, and a light sensor (TSL250R) that outputs an analog signal, with the voltage being directly proportional to the incoming light intensity. All of those components are attached in a 3D printed cuvette holder chamber (designed by Dr. Erik Gullberg), made specifically for this purpose.
Moreover, in the design of the custom fluorometer included two buttons and two extra indication LEDs. The function of the one button is to start the measurement that is about to take place, while the other button is used in order for the user to change mode. One of the available modes is the Comparison mode and the other one is the Timelapse mode. The indication LEDs are used to indicate which mode is currently running. The Comparison mode is used in order to compare several samples interchangeably, by inserting a new cuvette with a new sample each time and pressing the start button. The Timelapse mode is almost the same piece of code, with the difference that its running time and the intervals between each read are way longer than the ones in the Comparison mode. The Timelapse mode is useful when we want to measure the changes in the fluorescence intensity of one sample over time.
Below in the Figure 1, is data gathered from the Comparison mode representing the relative fluorescence of each sample.
Finally, the fact that we use an analog light sensor means that the output values are very sensitive to external or internal noise. For this reason the circuit should be as insulated as possible and the output data should sampled and filtered. Since our fluorometer is in a prototyping phase all this period, we were not able to have a very good insulated circuit. Thus, we really had the need to have a good sampling and filtering algorithm. So, we sacrificed some speed in order to capture more samples per measurement, and we also implemented a combination of a median and a mean filter in our sampling data. The median part of the Median-Mean filter, is modified to capture not a single median value, but several values around the median. Then, the mean part of the Median-Mean filter calculates the average of those median values, for the final result.
A picture of the actual construct is presented in Figure 2.
References
Schell, Mark A. "Homology between nucleotide sequences of promoter regions of nah and sal operons of NAH7 plasmid of Pseudomonas putida." Proceedings of the National Academy of Sciences 83, no. 2 (1986): 369373.
Curry, Robert E., Harry L. Pardue, Glen E. Mieling, and Robert E. Santini. "Design and Evaluation of a Filter Fluorometer That Incorporates a Photon-Counting Detector." Clinical chemistry 19, no. 11 (1973): 1259-1264.
Banzi, Massimo. Getting started with Arduino. " O'Reilly Media, Inc.", 2011.