We have continued our work on the project “Cloning, Expression, and Purification of the Cellular TORC Protein”. At the end of the fall semester we were working on troubleshooting the process of cloning the TORC2 gene into bacterial cells. We were working on testing the ligase by performing a single digest on both the insert and the vector with one of our enzymes. We saw colonies on the plates with and without ligase, but the plate without ligase has significantly more colonies. This indicates that the ligase does not have an effect and might even be causing a negative effect.
At the beginning of this semester we had to test our DH5-alpha cells to see if they were still competent because the freezer went down over the winter break and competent cells need to remain frozen when they are not being used. Several test transformation was performed with the cells and were all unsuccessful. New competent cells were made and tested with negative results. A second round of cells was successfully made and the cloning process began again. We performed a digestion on the pGEX vector, but we still had digested TORC2 inserts left from the previous semester. We used the digested vector and inserts in a ligation then followed up with a transformation to confirm that the ligase is not working. The results showed no colonies on any of the plates. We will continue to troubleshoot, but will probably need to buy new enzymes or ligase.
Dr. Isaacson had a successfully transformed TORC3 in the pGEX vector, so we moved onto the purification step for TORC3. We then took pGEX-TORC3 and transformed it into Rosetta DE3 pLysS and Rosetta DE3 pLacI competent cells, which should enhance expression of eukaryotic proteins in bacterial cells. We have started test inductions using IPTG to induce protein expression on the TORC3 wildtype and mutants. We have used a Coomassie stain and Western blot to test the TORC3 protein expression and are waiting on those results.
Moving on from here we will continue to try to clone the TORC2 DNA into the bacterial cells, but we will move on with the TORC3 expression step in the meantime.