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Team:TP CC-SanDiego/Project.html
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| - | 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 | + | 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 <a style="text-decoration:underline; color:white; font-size: 18px; background: none; padding:0px;" href="https://2014hs.igem.org/Team:TP_CC-SanDiego/Journal.html#linkheaderthree">Seamless Cloning</a>. 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. </p></div> |
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Revision as of 01:55, 21 June 2014
The Project Background and Explanation
AflatoxinB1-Detoxifizyme is produced by Armillariella Tabescens intracellularly. Its gene is analyzed to have no introns and has been recombined into a plasmid before. Extraction is rather difficult and commercialization has not been able to be done yet.
Zearalenone Hydorlase is produced by Clonostachys Rosea. Its gene is also analyzed to have no introns and has been recombined into a plasmid before. Similar to ADTZ, commercialization has not been done yet.
Natural E. coli secretes various proteins by signal peptides that recognize translocons. We focus on the SEC pathway that lets the protein fold in the periplasm first, as it usually results in a more accurate folding, correct formation of disulfide bridges, minimizes degradation, and is less likely to result in accumulation.
E. Coli Capable of Extracelluar Secretion of Mycotoxin Detoxifying Enzymes
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.
The Process to Complete our Project
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. 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.
Teamwork, education, and assistance
The TP_CC-SanDiego iGEM Team came up with the idea during its brainstorming meetings and performed the laboratory work. The team made group decisions about the direction of the project, troubleshooted, and put together the data and presentation.
The Simpson Joseph Laboratory of UCSD provided the students with the lab space, equipments and various reagents that allowed the students to perform the web laboratory work. The members of the Joseph Lab gave the team many tips and protocols. The Jeff Hasty Laboratory of UCSD provided the team and others with lectures and past iGEM experiences that helped them greatly.
Dr. Chang-Ho Baek from Thermo Fisher mentored the team throughout the project, gave them lectures, gave them advice for troubleshooting when there were obstacles. Thermo Fisher provided the team various reagent donations.
