The CasRx plasmid arrived in the DH5 alpha strain of E.Coli, which is suitable for DNA transformation, but not ideal for expression. In order to enhance the efficiency of expression, the solution used was to transfer the plasmid to the Rosetta BL21-DE3 strain, which is built for this task, and also aligns with our reference article [1]. After completing the Miniprep procedure, the plasmid will be isolated and thus, the transformation in the appropriate strain can be done, facilitating the expression of the protein.
As the quantity of the needed strain, Rosetta BL21-DE3, runned low in the lab, the iGEM team seized the opportunity to acquire new skills while preparing additional stocks. Those would not only be valuable for the team, but it will also benefit the whole laboratory staff.
Following the completion of Miniprep to obtain the plasmid, and the acquisition of competent Rosetta BL21-DE3 strains, the next step is to execute the transformation process, by transferring the plasmid into the previously talked expression strain. This step is crucial to obtain our protein and conduct the desired researches.
With the glycerol stock, our objective is to secure a long-term storage solution for the previously transformed strain. After cultivating and verifying its viability, the next step is to aliquot it into glycerol stocks. This preservation method ensures that, for future experiments, the strain will be available, and thus enhances at the same time the laboratory’s resources and our management of it.
Considering the experimental design, for both target and guide samples, a PCR step is essential in order to get complete sequences. Not only will it permit the production of testing materials at will, but also it will reduce the necessity to produce any orders in the future, ensuring availability of the genetic substance.
As the subject is synthetic sequences derived from a virus, the WNV, the approach aims to mimic as closely as possible the natural conditions. In order to achieve this, tests involving the conversion of these sequences into RNA were conducted, necessitating in the process a T7 transcription step.
The primary of the project being the virus detection through fluorescence, with the eventual goal of integrating this into a device equipped with optical measurement tools to detect the presence or not of pathogens. The so-said device would then relay the results to epidemiologists and hospitals, but first, the concept needs to be validated. To do so, in vitro experiments need to confirm the functionality of the detection method.
Conducting in vitro experiments represents the majority of the proof of concept part, but the project doesn’t end here. As the ultimate goal involves testing our detection method with mosquitoes, we sought collaboration with MIVEGEC, a research institute on viruses in Montpellier. Through this, we acquired our test subjects for evaluating the functionality, or not, of the detection in an in vivo setting.
Considering the focus of this study being a Cas protein not already available in the lab, the first part of this new experiment would be to express the protein from the transformed cells that previously integrated the ordered plasmid.
Producing the protein is but the beginning of this experiment, the ultimate goal being the utilization of the Cas. Consequently, a significant part of this project consists in actually extracting a purified solution of the protein from the cellular media. To do so, the purification process needs to be done in specialized columns for the Histidine tag and the MBP site, which are present on the target.