Team:UIUC Illinois/Project/Future

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<center><p style="font-size: 400% ">Puppy Probiotics.</p></center>
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<center><p style="font-size:60px">Future Directions</p></center>
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<h2>An Introduction</h2>
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<br><center><p style="font-size:30px"><i>"Biology has at least 50 more interesting years. "</i></p></center></br>
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<br><center><p style="font-size:15px">James Watson, 1986</p></br></center>
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<br><b>Imagine...</b></br>
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<br>
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  Imagine drinking a cup of coffee. You stolidly sip your espresso and soon enough you feel a warm sensation cajole your body into getting that assignment done. However, you notice you're fidgeting uncontrollably, the room starts spinning and you begin to vomit. Ambulances, the noises are familiar, you've just seized. You're a dog.  
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<h2>Environmental Remediation</h2>
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<br><b><p style="font-size:15px">Waste Matters</p></b></br>
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<p style="font-size:15px">Knowing that caffeine could be degraded, we wondered what became of the coffee plants that were used and abused for the world's most socially acceptable drug. Coffee production is no light matter. Large amounts of water go into coffee processing, and subsequently a large amount of waste is developed. "Commercial coffee is obtained from coffee
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cherries, 6% of which generate the coffee powder
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whereas the remaining 94% constitute the by-
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products such as husk, pulp water etc." [3]
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The massive waste production is not the only problem. It's easy to think the dry mass could be repurposed as a carbon & nitrogen source for animals and plants. However, reactions to stimulants such as caffeine are far reaching. It has been shown that caffeine can "inhibit seed
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germination and growth of seedlings"(Friedman
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and Waller 1983) as well as stunt the growth of cattle. By decaffeinating the waste, it can subsequently be used as feed and fertilizer and therefore have positive economical effects. Who knows, if coffee waste decreases and the coffee producers have an extra source of income, your next cup of coffee might be cheaper!</p>
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<br><p><center><img src="https://static.igem.org/mediawiki/2014/d/d2/CoffeeWaste3.jpg" alt="Smiley face" width="80%" height="450"></center></p></br>
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<br><p style="font-size:15px">To ameliorate this issue of waste, we looked to different pathways that could prove utile degrading xanthine waste. One organism, Pseudomonas putida CBB1 contained a set of genes that encoded for a caffeine dehydrogenase pathway. Instead of the N-Demethylation characteristic of our aforementioned puppy probiotic operon, we explored the caffeine dehydrogenase mechanism. In contrast to N-Demethylation, this pathway is a direct method to go directly to Trimethyl Uric Acid, another "safe" compound for the host animal who cannot degrade caffeine! We began to develop primers that would isolate the cdhA, B, and C genes, and moreso amplify out the entire operon, however our PCRs were unsuccessful.</p>
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<br><p><center><img src="https://static.igem.org/mediawiki/2014/7/7f/F3medium.gif" alt="Smiley face" width="400" height="200"></center>
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    <p><b>What just happened?</b>
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<br>Canines and many small animals are incapable of metabolizing theobromine and related stimulants as fast as humans can. Chocolate contains theobromine, an alkaloid that has stimulant properties. An excess of the stimulant builds up and begins to overstimulate the animal, thus leading to many malicious side effects. Ranging from slight nausea to death, consumption of these compounds are harmful for the animal. Chocolate can affect the animal regardless of the amount administered. Even the smallest amounts can produce side affects like diarrhea. In fact, theobromine has been employed to ward off pests of the same genus as canines: "Results indicate that mixtures of theobromine and caffeine have the potential to be developed into a selective, effective, and socially acceptable toxicant for the control of pest coyotes."[1] Caffeine in humans is degraded via a cascade mediated by Cytochrome P450. This is the primary molecule that allows for xanthine demethylation. In canines and smaller species, the cytochromes are available, however altered in a way that prevents caffeine degradation! Despite sequence homology to Human's P450, a canine's metabolism does not function similarly!
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<h2>Yogurt</h2>
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    <p><b><p style="font-size:15px">Let's get cultured.</p></b>
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<br><p style="font-size:15px">The final piece to our project aimed at propagating the demethylation proteins in some sort of food vector. We figured making yogurt via Lactobacillus fermentation was our best shot. It is widely known that yogurt is made through fermentation. Species such as Lactobacillus plantarum in yogurt break down lactose and produce lactic acid, the product which gives yogurt it's texture as well as taste. Having the culture that creates the yogurt simultaneously produce assistive proteins seemed like a clear shot to us!</p>
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<p><br><center><img src="https://static.igem.org/mediawiki/2014/a/a0/Lactobacillus.jpg" alt="Smiley face" width="350" height="350">
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<p><br><center><img src="https://static.igem.org/mediawiki/2014/8/8b/CDH_complete.PNG" alt="Smiley face" width="600" height="500">
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<br><i><p style="font-size:15px">This is the prospective construct of our CDH plasmid. With CP25, a near constitutive Lactobacillus promoter, we aim to produce the dehydrogenase gene excessively to degrade caffeine in polluted environments.</p></i>
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Latest revision as of 03:20, 18 October 2014


Future Directions


"Biology has at least 50 more interesting years. "



James Watson, 1986



Environmental Remediation


Waste Matters


Knowing that caffeine could be degraded, we wondered what became of the coffee plants that were used and abused for the world's most socially acceptable drug. Coffee production is no light matter. Large amounts of water go into coffee processing, and subsequently a large amount of waste is developed. "Commercial coffee is obtained from coffee cherries, 6% of which generate the coffee powder whereas the remaining 94% constitute the by- products such as husk, pulp water etc." [3] The massive waste production is not the only problem. It's easy to think the dry mass could be repurposed as a carbon & nitrogen source for animals and plants. However, reactions to stimulants such as caffeine are far reaching. It has been shown that caffeine can "inhibit seed germination and growth of seedlings"(Friedman and Waller 1983) as well as stunt the growth of cattle. By decaffeinating the waste, it can subsequently be used as feed and fertilizer and therefore have positive economical effects. Who knows, if coffee waste decreases and the coffee producers have an extra source of income, your next cup of coffee might be cheaper!


Smiley face



To ameliorate this issue of waste, we looked to different pathways that could prove utile degrading xanthine waste. One organism, Pseudomonas putida CBB1 contained a set of genes that encoded for a caffeine dehydrogenase pathway. Instead of the N-Demethylation characteristic of our aforementioned puppy probiotic operon, we explored the caffeine dehydrogenase mechanism. In contrast to N-Demethylation, this pathway is a direct method to go directly to Trimethyl Uric Acid, another "safe" compound for the host animal who cannot degrade caffeine! We began to develop primers that would isolate the cdhA, B, and C genes, and moreso amplify out the entire operon, however our PCRs were unsuccessful.


Smiley face


Yogurt


Let's get cultured.


The final piece to our project aimed at propagating the demethylation proteins in some sort of food vector. We figured making yogurt via Lactobacillus fermentation was our best shot. It is widely known that yogurt is made through fermentation. Species such as Lactobacillus plantarum in yogurt break down lactose and produce lactic acid, the product which gives yogurt it's texture as well as taste. Having the culture that creates the yogurt simultaneously produce assistive proteins seemed like a clear shot to us!



Smiley face



Smiley face

This is the prospective construct of our CDH plasmid. With CP25, a near constitutive Lactobacillus promoter, we aim to produce the dehydrogenase gene excessively to degrade caffeine in polluted environments.


The Institute for Genomic Biology
Biosystems Design, South wing, 2nd floor
University of Illinois at Urbana-Champaign
1206 West Gregory Drive
61801 Urbana, Illinois
illinoisigem@gmail.com
Copyright (c) 2014 igem.org. All rights reserved.

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