Difference between revisions of "Team:Munich/Measurement"

Although fluorescence measurements are a fundamental tool to characterize biochemical circuits, suitable measurement instruments are usually very expensive and therefore not available for all iGEM teams. We therefore constructed a portable low-cost fluorescence detector that can be assembled for less than 15$ and can measure time traces with the sensitivity of a commercial plate reader. The assembly of the detector only requires standard electronic components and a 3D printer. We provide equations to calibrate the data from the detector including a complete consideration of the propagation of uncertainties and wrote program code to automatize data analysis.

As a first proof of principle we measure a time trace of Cas13a digesting RNaseAlert with our detector. RNaseAlert is a commercially available molecular beacon consisting of a RNA linker, a quencher and a fluorophore, that get separated and therefore fluorescent when cut. For comparison, we also measured a time trace of the highly active RNaseA digesting RNaseAlert and a time trace of a sample containing only RNaseAlert. The time traces measured with our detector are shown in the figure below.

Time lapse measurement of Cas13a digesting RNaseAlert on paper using our detector. The positive control contains RNaseA and RNaseAlert. The negative control contains only RNaseAlert. Data points are connected with lines for the convenience of the eye. Error bars represent the measurement uncertainties of the detector.

The data show typical curves of enzyme kinetics. It can be seen that RNaseA is more active than CAS13a. This shows that our detector is in fact able to quantitatively measure different levels of enzyme activity and can therefore be used to characterize biobricks.

Overall Design

The conceptional design of our fluorescence detector is illustrated in the figure below. Light from a blue LED is filtered by a blue filter foil and excites fluorophores on a filter paper. The excitation light is blocked by an orange filter foil while the emission light from the fluorophores passes through the orange filter foil and illuminates a light dependent resistor (LDR). The LDR changes its resistance R_LDR corresponding to the intensity of the fluorescence light. Finally an Arduino Nano measures the resistance via a voltage divider and calculates the fluorophore concentration.

Overall design.

Framework of equations for Calibration and data Analysis

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