Cloning, characterization and expression of A. thaliana G protein α-subunit gene for structural studies

A combined PCR-cloning strategy was implemented to express GPAl for structural studies. To begin with the presence of GPAl, which İs the ?-subunit of heterotrimeric G proteins from Arabidopsis thaliana, was checked with restriction enzyme digestion and sequence analysis. PCR was done on the pCIT857 vector including the coding sequence of GPAl. Appropriate primers were designed with and without the restriction enzyme sites to clone the GPAl into both subcloning and expression vectors. Once the above objective was analysed, three different parallel approaches were used to clone-GPAl from the pCIT857 vector into the expression vectors. In the firstapproach, GPAl was subcloned into several vectors including pGEM® -T Easy (Promega), pCR® II- TOPO (Invitrogen), pCR® -XL-TOPO (Invitrogen) vectors. Then GPAl derived from these vectors were cloned into expression vectors containing PGEX-4T2 (Amersham Pharmacia), pGFPuv (Clonetech), pETM-11 and pETM-30 (EMBL, Heidelberg). In the second approach, GPAl from the pCIT857 vector was directly cloned into the expression vectors. Different fusion partners of expressed GPAl, GST in pGEX-4T2 and GFPuv in pGFPuv vector and His-taq in pETM vectors, were used to yield the most efficient expression of GPAl, that would form the basis of subsequent structural characterisation experiments.In the third approach, GPAl from pCIT was directly cloned into the expression vectors including pCR® T7/NT TOPO (Invitrogen) and pTrcHis® TOPO (Invitrogen) vectors. Of the three approaches, this one proved most convenient as the number of steps and manipulations was limited in comparisons to those above. During all subcloning and cloning steps, different strains of E. coli including XLlBlue, TOP10 and TOP10 F' were used as hosts for subcloning and BL21(DE3), BL21(DE3)pLysS, Rosetta(DE3), Rosetta(DE3)pLysS and BL21-CodonPlus® (DE3)-RIL were used for expression. The rationale being that different strains often perform differently.To summarise GPAl was successfully cloned into 3'MCS of pGFPuv, pCR® T7/NT TOPO (Invitrogen) and pTrcHis® TOPO (Invitrogen) vectors. In particular the verification was based upon the restriction enzyme digestion and sequencing data. Also the recombinant protein was expressed both using different IPTG concentrations and temperatures. But the expression was not detected by SDS-PAGE analysis. Recombinant protein is currently being expressed and further work is in progress to improve the properties of bacterial expression system

Reflections on a pandemic-related disruption to a preparing future faculty pilot program in Turkey

University faculty members simultaneously hold multiple roles in teaching, research and community. One way of preparing graduate students for these roles is through the Preparing Future Faculty program that is commonly found in the US universities. There is, however, little support in Turkey to prepare graduate students for faculty roles and responsibilities. To address this gap, two mid-sized universities in Turkey modified a Preparing Future Faculty program for the Turkish context. This flagship program was established to focus on teaching and community service since the results of focus groups conducted with graduate students at the two universities primarily indicated interest in these areas. In this paper, we reflect on the initial implementation of this program, the changes made to move this pilot program online and students’ experiences with this new mode. We make recommendations for benefitting from these changes in the resulting program