We began working in our brand-new lab space! When we first arrived, the lab was nothing but two work benches. We had to collect and organize the equipment, materials, cell culture room, and work area; all according to lab safety standards. We also began cloning the plasmids for our experiments and learning how to work with cell culture by practicing on HEK293 cells.
Highlights: clone Steap4, Timp1, F4/80 into pGL4.17 and pGL3 plasmid for Promoter Assay.
In March, we experienced our first little success. We cloned our promoters into the pGL3 vector and were able to begin our first experiment, the Promoter Assay. Meanwhile, so far, our practice cells were alive and well.
Highlights: A decision was made to concentrate our efforts on the pGL3 plasmid expression assay. Because, then we could compare the expression among the promoters and we wouldn’t have to use two sets of luciferase kits.
Our long-awaited microglia and astrocytes arrived! While our cell lines were getting used to their new home, we began cloning our crisper and gRNA plasmids for our two-component system.
Highlights: Received microglia and astrocytes cell lines, px601, H1, H2 CMV-dCas9-VPR, building plasmids with Gibson assembly.
After a tireless journey, we successfully cloned most of our system’s plasmids. Now we began attempts to transfect these plasmids into our cell lines with different transfection reagents. Additionally, we began the promoter assay and astrocyte reactivity assay.
Highlights: Began Promoter assay, transfection attempts with reagents, ELISA for astrocyte reactivity assay.
In June we had to face the music; our transfection was not working. We began exploring alternatives ways to insert our plasmids into our cells, such as electroporation, calcium phosphate transfection, and additional transfection reagents.
Highlights: Astrocyte Western blot, Electroporation of microglia.
In July we preformed further attempts to transfect our cells and reactivity assays, and let’s not forget exams period.
Highlights: Electroporation of microglia, mycoplasma assay, colony PCR after the microglia electroporation, transfection attempts with reagents, astrocyte reactivity assay using western blot.
The summer was in full swing and with it our iGEM team. Each team member worked full force on experiments to reach a proof of concept.
Highlights: continued reactive astrocyte experiments.