Perspective

Checking how the multiplexing can work, whether multiple targets can be put in the mix with only 1 guide and see how the fluorescence will react (and thus, checking if there are some off-target effects on whole sequences).

Checking, on the other side, the impact on fluorescence levels of having multiple guides and one target in the same reaction. Moreover, investigating if it is possible to have multiple guides and their respective targets in one reaction could provide useful informations about the behaviour of the SHERLOCK system, and how one could push its limits further. Multiplexing detection in one single reaction would greatly simplify and speed up the detection process in the final machine application of our project and guide the design of microfluidics features.

Protein Purification

As discussed before, we did not manage to purify CasRX. There are a few things that can be elaborated. Firstly, it is interesting to test the expression in different strains, other than E.coli Rosetta BL21(DE3), but classic BL21 E.Coli. The point isoelectric (pI) of our protein containing all tags is at 7.5, so it is possible to test other buffers composition, as long as it is one pH unit , whether it is below or above 7.5. Our protein came very contaminated out of the Ni-NTA column, so it is necessary to add a Ni-NTA reverse step, after the His tag is cut with the TEV enzyme.

Expression

Test another strain, E.coli BL21 which is largely used for protein expression. Test auto inducible ZYM medium instead of Terrific Broth, which would avoid putting cytotoxic IPTG in the medium.

Lysis

Try another lysis buffer composition and different sonication intervals for a better cell lysis.

Purification

try another wash and elution buffer composition and pH, it should be correct as long as it is 1 pH unit different from protein pI (7.5 for CasRX before tag cleavage). For the Ni-NTA step, it should be good to test different elution steps and Imidazole gradients to get a better elimination of non-specific interactions and a cleaner product. If collected fractions are still contaminated, even after 4% Imidazole step during the Ni-NTA purification, we can add another step, which could be: after the Ni-NTA and cleavage by TEV enzyme steps are done, it can be useful to do the Ni-NTA reverse to assure the absence of all non specific interactions. Everything that does not have an His tag will be on the column, meanwhile CasRX will go into the flowthrough and it should be clean, because all these contaminants are now supposed to be caught by the column, as it should happen during the Ni-NTA purification.

Test Cas13a with off target effects to check how it affects the fluorescence (mutations) on different sequences (the main sequence 1 and the main sequence 2). To do so, it is possible to induce mutations on 1 or more nucleotides on the target, and use those new modified sequences to check what are the effects of using sequences that won't be perfectly the same and will have some nucleotides different that won't bind with each other.

In order to further optimize the gel electrophoresis, modifying one parameter did not get through analysis, as the agarose type used was not the most optimal one. Considering that the NuSieve is specialized in small bands, it might give better results, leading to better analysis of the so-said results and better representation in the end.

During our discussions with Pritam Saha, a Master's thesis student in Dr. Manouk Abkarian's lab (CBS, Montpellier), we gained valuable insights into the fields of microfluidics and machine development.

Our initial step would involve creating a blueprint for the machine, utilizing AutoCAD/Fusion 360 software. In the design, we envisioned the machine having four distinct compartments, each dedicated to specific functions: one for guideRNA and target substances, a second for the enzyme, a third for sensor molecules, and a fourth for chemical amplification. To facilitate amplification, we decided to incorporate a hot plate beneath the chambers, enabling isothermal reverse transcription PCR (RT RPA) and transcription processes.

Ensuring precise control over the flow of chemicals between compartments was a priority, and to address this, we conceived the idea of implementing small storage units behind each compartment. These units would be equipped with regulated sphincters to control the transfer of chemical quantities.

Furthermore, we planned to establish IoT connectivity, allowing epidemiologists and researchers to access results directly.

This phase of the project necessitates collaboration with experts in electronics, hardware, and computer science to bring our innovative machine to fruition.

As the detection in the mosquitoes did not work, a lot of hypotheses on these results emerged. The first one being whether or not viral RNA was present in the initial samples, which can be sorted out according to our supplier, which said “85 to 90% of the mosquitoes are infected with WNV”.

The next step would be to check if the target is mutated on the specific site of the binding, and it would be interesting to send some samples for sequencing.

After that, the RNA might be present, but degraded. This can be verified using a BioAnalyzer, considering that one is present in Montpellier at GenomiX, which might provide useful insight on the state of the RNA using (more information can be found about bioanalyzer on this website).

Finally, another reason of the missing output for the detection might be that the initial viral charge was too low, meaning that doing an amplification could solve this problem, for as long as sequences can be found in all the variants of the virus, sequences that can be found using the software designed by our team in the Software section of the wiki. But doing a normal PCR isn't enough to amplify viral RNA. To do so, it is mandatory to do a RT PCR (reverse transcription PCR) to change from RNA to DNA, and only after that, it is possible to amplify the fragments and get more viral DNA. It also means that another step of transcription is needed afterwards, to go back to RNA samples and do the detection.