Wet Lab Notebook
Learn continually - there's always "one more thing" to learn!
Our journey in the laboratory started in May 2020. Before that, knowing that it was possible to deal with site-directed mutagenesis and eukaryotic cells, the wet lab team's members were trained under the guidance of our Instructors. The purpose of the training was to get familiarized with basic experimental protocols and techniques in synthetic biology, such as ligations, digestion, transformations, and transfections. An essential part of the training was the biosafety instructions that ensured the unmolested of the experiments. During the training, the mock-tests were performed using water instead of actual reagents multiple times until they were fully prepared to successfully and safely conduct the experiments.
Meanwhile, we conducted a literature search for the targeted genes, CYP2D6 & CYP2C19, the drug categories metabolized by them, and their involvement in the personalized therapy. Also, we managed to purchase the initial wild-type templates for CYP2D6, CYP2C19, CPR, and cytochrome b5 cDNAs. Finally, before starting the site-directed mutagenesis, the design of primers needed for this procedure was carried out.
Due to the COVID-19 pandemic, the start of our wet-lab experiments and training was delayed until April.
In April 2021, the training above took place in the Laboratory of Applied Mechanics and Oscillations with our instructor Diana Portan and in the Laboratory of Biology in the School of Science and Technology in Hellenic Open University, under the supervision of the Associate Professor Argyro Sgourou.
In May 2021, the first experiments were performed. Firstly, amplification of the cDNA fragments used in our methodology was conducted. The next thing we dealt with was the TOPO directional cloning of pENTR/D-TOPO vectors with the wild-type CYP2D6, CYP2C19, CPR, and cytochrome b5 cDNAs. Finally, this month we went on with the procedure of transforming High-Efficiency E.coli cells with these vectors.
In June 2021, we had to deal with the plasmid DNA extraction procedure. After we achieved this, we managed to perform site-directed mutagenesis in the plasmids extracted and transform the bacteria with them.
In July 2021, our wet lab members performed the digestion of mutated cDNA fragments of CYP2D6 and CYP2C19, which then subcloned into mammalian vectors.
In August 2021, we proceeded to the next step: transfection of the mammalian expression vectors in eukaryotic cells. Finally, we proceeded to the isolation of the microsomal protein fractions. At this point of our experiments, the transfected cells were sent to our partner Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences at Tohoku University, Japan
In September 2021, we were waiting for the results from the UPLC for the CYPD26 variants and the LC-MS/MS for the CYP2C19 variants and preparing our Wiki Page.
In October 2021, we finally obtained the results needed for our project and started their Interpretation.