Team:UPF Barcelona/Wetware testing

ARIA

Wetware

Biosensor library testing

Testing the biosensors is the third part of the engineering cycle stage. In the following page, we expose the steps followed to carry it out.

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 [1], 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.

This picture represents the activation of the fluorescent signal. At the left it shows the inhibition of the fluorescent reporter by the quencher, whereas at the right it shows how the active Cas12-gRNA complex cuts the fluorescence reporter, keeping the fluorophore separated from the quencher, thus able to emit fluorescence.

Figure 14: Fluorescent Signal and Cas12 Cleavage.

For our fluorescent measurements we used the Tecan Infinite 200 PRO plate reader [2]. 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 [3].

The picture shows at the top a plate with several wells that contain different samples. At the bottom of the image the plate is introduced into the plate reader.

Figure 15: Fluorescence Measurements.

To be able to use the plate reader we had to follow the Instructions of Use for i-control plate reader [4]. For the parameterization of the plate reader we did it as follows:

Parameters	Value
Temperature	37ºC / Room temperature
Duration	2h
Use kinetic interval	Measure every 5 minutes
Excitation	530 nm
Emission	560 nm
Number of flashes	20
Measure from	Bottom
Gain	Optimal (100 RFA) + Use Gain Regulation

Experiments replication

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.

IPTG induction

It is a method of regulating DNA transcription thanks to T7 polymerase expression regulation [5]. We have used it for inducing the gRNA and LbCas12a protein transcription. Specifically, the optimal IPTG concentration used was 100mM.

Protocol

At the top of the image there are two eppendorfs. One is full of antibiotics and the other one contains IPTG. The two elements are introduced into a 15mL tube with liquid culture.

Figure 16: IPTG Induction and specific antibiotic.

Lysozyme lysis

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 [6]. We have used this method to release the Cas12a protein and the gRNA that allow the specific antibiotic resistance gene sample detection.

Protocol

In the upper left part of the picture we can have a 50mL tube filled with LB-culture. Then, this tube is centrifuged and in this way we obtain a pellet of the cells. To the obtained pellet we add the lysis buffer and resuspend it. The resuspended solution is transferred to a 1,5mL tube and the Lysozyme solution is also added to the 1,5mL eppendorf. Next, it is incubated and centrifuged and ready for the next step: detection.

Figure 17: Lysozyme lysis scheme.

Plate Reader 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.

At the left there is a 1,5 mL eppendorf tube, representing all possible reagents to be added. They are pipetted into the wells of a plate for the plate reader. After that, the plate is introduced into the plate reader and then the fluorescence intensity is measured.

Figure 18: Detection of resistant genes in the plate reader.

Results Analysis

After having measured the fluorescence intensity in the plate reader, we’ve analysed our results by making plots and discussing them with the team.

In the left part of the image there are two graphs showing the results obtained from the plate reader. On the other side, we have the team discussing and analysing the results.

Figure 19: Analysis and discussion of the results obtained from the plate reader by ARIA wet lab team members.

Autolysis testing: Bradford's assay

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) [7].

Protocol

Representation of the Bradford Assay. Scheme of colorimetric change from brown to blue after the addition of a protein complex, quantitative measurement and extrapolation of absorbance to a BSA standard curve.

Figure 20: Bradford Assay.

References

[1] 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

[2] Ag, T. T. (n.d.). Plate reader Infinite 200 PRO. Retrieved October 5, 2021, from: Tecan

[3] 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

[4] Instructions for the use of iControl (n.d.). Retrieved October 5, 2021, from: iControl

[5] How does IPTG induction work? (n.d.). Retrieved September 26, 2021, from: Goldbio

[6] Shehadul Islam, M., Aryasomayajula, A., & Selvaganapathy, P. (2017). A review on macroscale and microscale cell lysis methods. Micromachines, 8(3), 83. doi: 10.3390/mi8030083

[7] Bradford Assay (Bradford Reagent) - ES. (n.d.). Retrieved October 19, 2021, from: Thermofisher