Biosensor library testing
The aim of this wetware part is to test if the Alexandria Library Biosensors are able to detect specifically if a sample is resistant or not to one antibiotic. For this, the built biosensors were tested with isolated plasmids containing an antibiotic resistance gene corresponding to each of the gRNAs.
Detection and reporter functioning
Our biosensors are based in the CRISPR-Cas system. If the target sequence is present in the analyzed sample, the gRNA-Cas complex gets activated and cleaves the target sequence, but also enables transcleave collateral activity, thus cutting all DNA around , including the reporter sequence, that is also in the media. When the cut occurs, the fluorophore is separated from the quencher and the fluorescence signal, which was previously inhibited by the quencher, is activated. As a consequence, we are able to measure the fluorescence of each sample in the plate reader and determine the resistance or not of our sample to a specific antibiotic.
For our fluorescent measurements we used the Tecan Infinite 200 PRO plate reader . The operation of the Plate Reader is based on a pulsed excitation light, at a specific wavelength, that is emitted by a laser source, and the fluorescence emissions of the samples. First, excitation light is focused through a pinhole to the center of each well of a microplate and after this step, fluorescence emission is collimated by a parabolic mirror and its decay is digitized and acquired for analysis .
To be able to use the plate reader we had to follow the Instructions of Use for i-control plate reader . For the parameterization of the plate reader we did it as follows:
|Temperature||37ºC / Room temperature|
|Use kinetic interval||Measure every 5 minutes|
|Number of flashes||20|
|Gain||Optimal (100 RFA) + Use Gain Regulation|
For testing the Alexandria Library Biosensors a few steps need to be done: Inducing the gRNA and Cas12 expression, Lysing the cells, and using the plate reader for reading the fluorescence emitted by the reporter once cuted by the Cas12. After this, the obtained results must be analyzed. A small test about the autolysis approach has also been included in this section.
It is a method in which the cell membrane is broken down in order to release intra-cellular materials such as DNA, RNA, protein or organelles from a cell . We have used this method to release the Cas12a protein and the gRNA that allow the specific antibiotic resistance gene sample detection.
The plate reader allows to determine the fluorescence intensity of the samples and define if there has been a positive or negative detection. We’ve used it to determine if our samples had presence or absence of specific resistance genes. The reporter kit protocol was used to prepare the reactions to be detected. The detection conditions and obtained results are discussed in the results section, and the optimal experiment conditions are explained wetware proof of concept page.
After having measured the fluorescence intensity in the plate reader, we’ve analysed our results by making plots and discussing them with the team.
One of the simplest and cheapest ways to visually and quantitatively verify that cells have been lysed is by Bradford's assay. It is a colorimetric assay based on a dye called Coomassie Brilliant Blue G-250, that can bind protein complexes. In an acidic environment and after the addition of the proteins, there is a shift from the reddish-brown color of the dye to its bluish form. This change can be observed at naked eye or by measuring the absorbance at 595nm with a Spectrophotometer. Finally, to quantify the amount of protein in the compound, the measurements can be extrapolated to a BSA standard curve (blank) .
 Schindele, P., & Puchta, H. (2020). Engineering CRISPR/LbCas12a for highly efficient, temperature-tolerant plant gene editing. Plant Biotechnology Journal, 18(5), 1118–1120. doi: 10.1111/pbi.13275
 Petersen, K. J., Peterson, K. C., Muretta, J. M., Higgins, S. E., Gillispie, G. D., & Thomas, D. D. (2014). Fluorescence lifetime plate reader: resolution and precision meet high-throughput. The Review of Scientific Instruments, 85(11), 113101. doi: 10.1063/1.4900727