Team:TP CC-SanDiego/Project.html

Our idea is to attach signal peptides to the N-terminus of the detoxifying enzymes. We use alpha amylase signal peptides and beta-lactamase signal peptides. The original proteins for these signal peptides are



secreted out naturally by E. Coli, especially in strain K that we use for our project. They both follow sec-dependent pathways. In the case of the beta-lactamase signal protein, cases of successful fusion proteins secreted by the



beta-lactamase signal protein have been shown. Successful continual excretion of the mycotoxin degradation enzymes can be purified in mass amounts periodically, or the chimeric E. Coli can be placed directly on



crops to prevent and combat mycotoxin exposure. Since there is no current cure to mycotoxin exposure, enhanced prevention with successful secretion will be very influential.

In order to achieve our goal, we modified our vector and synthesized genes with reverse PCR and primers with specific tails. After the parts were assembled by Seamless Cloning (Link to the Protocol page of our wiki). The parts were transformed in an LB+Amp agar plate, and colony PCR was performed for an initial verification step. After the initial verification, the plasmids were purified from the colonies that were declared positive and compared to the theoretical sequences. The final colonies were then grown, and each culture was separated into two - IPTG induced, and non-induced. The log-phase, induced and non-induced cultures were then separated into cell pellets and supernatant. SDS-PAGE gels were run for each induced pellet, non-induced pellet, induced supernatant, and non-induced supernatant for comparison. Induced and non-induced were compared to verify the functionality of the vector, and the pellets and supernatant were compared for excretion. The whole expression and secretion were verified by Western Blotting with anti-his antibodies.