Team:TP CC-SanDiego/Project

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<h1>Project Description</h1>
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Our project focuses on two main points:</div></p>
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1) The breakdown of deadly mycotoxins into harmless byproducts through the use of  various enzymes.</p>
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2) Maximizating production rates of detoxifying enzymes . </p>
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Mycotoxins are a type of harmful, and sometimes lethal, toxin produced by fungi. These fungi readily colonize improperly stored crops like maize, coffee beans, peanuts, pistachios, and tobacco. Consequently, mycotoxicosis, or poisoning from the consumption of mycotoxins, has become a prevalent problem in third world countries where crops are commonly improperly stored in moist conditions. Mycotoxicosis is also spread through the consumption of infected animals, including but not limited to, pigs, cows, and birds. </p>
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One notable mycotoxin is Aflatoxin B1 (AFB1), a potent natural carcinogen and an acutely toxic agent synthesized by Aspergillus flavus. 5-50 mg of AFB1 per kilogram of body mass will kill a median member of the human population, as grimly proved in Kenya, where the development of A. flavus on maize killed 120 local people and affected hundreds more in 2003. </p>
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Another widespread mycotoxin is Zearalenone, which is secreted by fungi of the Fusarium genus, including F. graminearum and F. cerealis. Zearalenone affects many cereal plants, including barley, oats, wheat, rice, and maize. Although the Zearalenone toxin (ZEN) is not as lethal as AFB1, it also functions as an oestrogen. Research has shown high levels of ZEN causes birth defects and interference with ovulation, implantation, and fetal development.</p>
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Thankfully, there is a solution to this dangerously extensive problem. Through research we found multiple mycotoxin-degrading organisms that secrete various detoxifying enzymes. These organisms, Armillariella tabescens and Clonostachys rosea, produce Aflatoxin-Detoxifizyme (ADTZ) and Zearalenone Hydrolase (ZHD101), respectively. ADTZ’s method of degradation of AFB1 is as follows: AFB1 is first converted into AFB1-epoxide and then hydrolyzed to form dihydrodiol. The final hydrolysis opens the difuran ring in dihydrodiol, ending the conversion of AFB1 into a vastly less dangerous form. The method of degradation used by ZHD101 on ZEN is unclear to researchers as of now, but it is hypothesized that the ZEN molecule is converted into a less oestrogenic form through a simple cleavage. </p>
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While these detoxifying enzymes are successfully synthesized by the fungi, secretion could be vastly improved by producing these enzymes in high-secretion bacteria such as E Coli. By creating a bacterial plasmid containing the signaling sequence (-lactamase or α-amylase), the gene of interest (ZHD101 or ADTZ), sfGFP (a super-folding variant of the classic Green Fluorescent Protein), and a HIS tag (for protein detection), the synthesized protein would be easily secretable, detectable, and isolatable. The increased production system using E. Coli will allow large quantities of detoxifying enzymes to be harvested for agricultural application. These mass-produced enzymes can then be used to destroy AFB1 and ZEN present on crops, reducing cancer incidences and deaths from mycotoxicosis. Perhaps, with the detoxifying enzymes that will be produced in our experimentation, the deadly outbreaks of these deleterious mycotoxins will be an incidence of the past.</p>
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        Background<br />
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    <span style="font-family: 'HelveticaNeueLT Pro 25 UltLt'; font-size: 18px;">The Project Background and Explanation</span>
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            AflatoxinB1-Detoxifizyme is produced by <a style="text-decoration:underline; color:black; font-size: 18px; background: none; padding:0px;" href="http://www.ncbi.nlm.nih.gov/pubmed/21239152">Armillariella Tabescens</a> intracellularly. Its gene is analyzed to have no introns and has been recombined into a plasmid <a style="text-decoration:underline; color:black; font-size: 18px; background: none; padding:0px;" href="https://www.legionpatent.com/patents/7695751/">before</a>. Extraction is rather difficult and commercialization has not been able to be done yet.
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            Zearalenone Hydorlase is produced by Clonostachys Rosea. Its gene is also analyzed to have no introns and has been recombined into a plasmid <a style="text-decoration:underline; color:black; font-size: 18px; background: none; padding:0px;" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1222652/">before</a>. Similar to ADTZ, commercialization has not been done yet.
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            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 more correct folding, correct formation of disulfide bridges, minimizes degradation, and is less likely to result in accumulation.
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        Idea<br />
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    <span style="font-family: 'HelveticaNeueLT Pro 25 UltLt'; font-size: 18px;">E. Coli Capable of Extracelluar Secretion of Mycotoxin Detoxifying Enzymes</span>
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    <div id="third-text">  Our idea is to attach signal peptides to the N-terminus of the detoxifying enzymes. We use alpha amylase <a href="http://www.signalpeptide.com/index.php?sess=&m=listspdb_bacteria&s=details&id=37&listname=">signal peptides</a> and
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    beta-lactamase <a href="http://www.signalpeptide.com/index.php?sess=&m=listspdb_bacteria&s=details&id=34&listname=">signal peptides</a>. 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 beta-lactamase signal protein, cases have been shown of successful fusion proteins secreted by the beta-lactamase signal protein (link to: http://www.ncbi.nlm.nih.gov/pubmed/6607473). 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 </div> 
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<div id="header-text">
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        How<br />
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    <span style="font-family: 'HelveticaNeueLT Pro 25 UltLt'; font-size: 18px;">The Process to Complete our Project</span>
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    Project 1 - 9
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    SPONSORS
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Latest revision as of 21:45, 19 June 2014

iGEM San Diego - Project

Background
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 more correct folding, correct formation of disulfide bridges, minimizes degradation, and is less likely to result in accumulation.
Idea
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 beta-lactamase signal protein, cases have been shown of successful fusion proteins secreted by the beta-lactamase signal protein (link to: http://www.ncbi.nlm.nih.gov/pubmed/6607473). 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
How
The Process to Complete our Project
Project 1 - 9

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