Part:BBa_K1519124:Experience
Bba_K1519123, the secretion-based purification vector, was characterized by the 2014 Michigan iGEM Team. To characterize the efficacy of the construct, we used two different methods. First, we created part Bba_K1519124, which is Bba_K1519123 with the Bba_J06505 MCherry reported ligated into the construct. This ligation allowed us to use epifluorescence microscopy to characterize the expression of our inducible purification construct. After transforming our construct in BL21 E. coli , we obtained fluorescent microscopy images of cultures grown overnight in the presence and absence of 100ug/ml IPTG.
From the microscopy experiment, we were not able to reach many conclusions. Clearly, our construct was inducing in the presence of IPTG and had standard leakiness in the promoter, resulting in some expression in the absence of IPTG. However, the resolution of epifluoresence made determining whether or not our construct was secreting the protein into the supernatant of the cells very difficult when the cells were highly induced. There was a halo of red surrounding the cells, suggesting that some of the protein may have been secreted from the cells. To further test the levels of secretion of the construct, we used a more easily observed approach.
Because the construct contains a polyhistidine tag, we were able to use western blots against the polyhistidine tag to approximate the levels of secreted construct in different cellular fractions. Western blot analysis of the whole cell lysate and supernatant indicated that our protein was secreted into the media of the cell when induced with IPTG. There was degradation of our construct in the induced lanes, likely due to tail-specific protease activity on the LVA tag of Bba_J06505. We accidentally incorporated the Bba_J06505 MCherry instead of Bba_J06504, resulting in an LVA tagged protein fusion and ultimately degradation of the product.
