Introduction
Designing repeatable measurement approaches, for use during the characterisation of new parts, allows results to be compared accurately to those in labs across the world.
This page describes how the photos taken of luminescent samples were analysed, and how data from the plate reader was used to obtain values with units of meaning. It also describes the development process of the custom measurement technique that was developed in the process.
Calibration of Fluorescence: Plate Reader
Our thermal pathway, TcI, produced eYFP when sucessfully activated. Calibrating fluorescence signals on a plate reader is a standard protocol and well documented. We followed the iGEM calibration protocol for plate reader fluorescence to calibrate our values. While our reporter protein was yellow, from comparing the fluorescein spectra to eYFP emission spectra, we thought that fluorescein would still be appropriate.
Calibration of Fluorescence: Camera
While plate reader data was beneficial in the early stages of the project, validating our concept required demonstrating the spatial activation of cells. Since spatial activation must be performed on a plate, plate reader measurements became unsuitable for this purpose. Therefore, we developed and integrated a camera imaging setup, including appropriate filters, into the MagentaBOX. This setup enabled real-time imaging of plates as they were magnetically activated, allowing us to observe activation as it occurred.
To ensure the camera setup produced relevant and accurate data, it was necessary to calibrate both the camera and the photo analysis system using standard solutions. We photographed a serial dilution of fluorescein and analysed the images using ImageJ (Fig. 2).
Method
Nine wells were filled with different concentrations of fluorescein using a serial dilution technique. These wells were imaged with the MagentaBOX, and the camera settings were saved for future replication. ImageJ was used to take repeated measurements, and the average mean grey value for each well was calculated. These values were then normalised to water and plotted against the fluorescein concentration (Fig. 3).
Calibration of Chemiluminescence: Plate Reader
For our EPG pathway, we used NanoLuc as the reporter protein. NanoLuc is a small, highly sensitive enzyme that catalyses the oxidation of coelenterazine, producing bright visible light. This is a reaction that emits chemiluminescence. Reasons for why this reporter system was used are described in our design page.
To compare our results with external results, we normalise the data obtained from our plate reader given in arbitrary units, to the emission of light from the oxidation of luminol catalysed by varying concentrations of copper sulphate. Unlike fluorescence, chemiluminescence measurement calibration protocols are not widely used and documented. We developed our own calibration protocol, building off of work done by the 2021 Marburg iGEM team.
Luminol normalisation
In a basic environment, luminol is oxidised in the presence of catalytic copper sulphate, emitting light. With the oxidising agent in excess and concentration of luminol constant, changing the concentration of copper sulphate changes the amount of light emitted. This reaction is cheap and reproducible, which makes it ideal for use as a reaction to normalise our data to.
See the custom chemiluminescence calibration protocol.
Calibration of Chemiluminescence: Camera
As with fluorescence, we also wanted to calibrate the camera system so that it could measure chemiluminescence. However the camera was not sensitive enough so we could obtain chemiluminescence data from the images.
However, we tried to use a phone camera with well adjusted low light condition, which successfully captured the brightest calibrant we have.
We tried to use the camera we retrieved to measure luminescence, but it turn out to be completely dark. This could probabily because the camera model we picked was not sensitive enough, or the photo-stackig algorithm we used is not enough to read the low light level of chemiluminescence.
Calibration of Chemiluminescence: Custom Hardware
We tried to carry out calibration for chemiluminescence on our photodiode OPT101. We set the gain to highest level using 50M ohm resistors, and completely block the light using our hardware
Unfortunately, the result we got is full of noise.
We tried to use luminol solution to calibrate the photodiode, but the noise is too strong that we cannot get any meaningful reading that can supress noise.
References
https://2021.igem.org/Team:Marburg/Measurement
Burgis M, Luminol Calibration. dx.doi.org/10.17504/protocols.io.bzdfp23n