To verify our construct was being produced by the cells we used fluorescence microscopy to detect the mCherry protein tagged to our construct. As we can see in the figure below, after 10 ms of red light exposure, the induced cell clearly emits a lot of light whereas the uninduced cell barely emits any light (the slight light emission they produce is due to promoter leakage). In essence, our construct is emitted upon induction.

To verify our construct was being secreted into the media we performed a western blot in which we detected our construct with an anti-polyhistidine antibody. As we can see in the figure below, when induced, our construct is being produced by the cells (induced pellet) and is being secreted into media (induced 40 uL supernatant). The presence of the construct in the uninduced cells could be due to promoter leakage. In essence, our construct is successfully secreted into the media.

Between the wiki freeze and the Jamboree in Boston, we plan on finishing the cloning of the antibody scFv in our purification construct, and provide evidence for antibody scFv function.

We will over express DADH (the antigen) in E.coli BL21 and lyse the cells. A sample of the lysed cell will be run on an SDS-PAGE gel and we will bind the protein to a nitrocellulose membrane.
Meanwhile our construct containing the antibody scFv will be overexpressed in E.coli BL21. Once harvested the media will be spun down and the supernatant containing our secreted construct will be collected.

We will incubate the nitrocellulose membrane with the collected supernatant overnight for our antibody scFv to bind to DADH. The nitrocellulose membrane will then be incubated with the anti-Myc antibody overnight. Lastly, we will incubate the membrane with a probed antibody that targets the anti-Myc antibody. If we can identify the probe on the nitrocellulose membrane then our antibody functions properly since it has successfully bound DADH (see figure below).

To compare our construct to the traditionally used pelB tag, we will perform the same methods we used to harvest, bind and identify bound scFv to DADH; this time using the pelB construct instead of our engineered construct. By comparing the intensity of the probe on the western blots of the two secretion systems we will be able to determine which tag produced the greatest amount of functional antibody scFv.

Be prepared for exciting results!