Team:UIUC Illinois/Results

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<center><p style="font-size: 400% ">Results</p></center>
<center><p style="font-size: 400% ">Results</p></center>
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<br><center><p style="font-size: 150% "><i>"I have had my results for a long time: but I do not yet know how I am to arrive at them. ~Karl Friedrich Gauss"</i></p></center></br>
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<br><center><p style="font-size: 150% "><i>"I have had my results for a long time: but I do not yet know how I am to arrive at them."</i></p></center></br>
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<br><center>-James Watson, 1986</br></center>
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<br><center>Karl Friedrich Gauss</br></center>
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<br>
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<br><h2>Feeding Assay</h2></br>
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Experimental analysis began with verifying biobrick BBa_K734000. To verify growth from solely caffeine or theobromine, the organisms were sent to us from UT Austin with the guaB gene knocked out. Guanine comes from xanthine, which is a necessary factor for microbial growth. Without the ability to produce guanine, xanthine degradation is imperative for guanine biosynthesis therefore addicting the microorganisms to caffeine. We set out experimentally to show that theobromine degradation could also be used for xanthine degradation using the same N-Demethylation pathway. Our results are as follow:
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<h2>Environmental Remediation</h2>
 
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<br><b>Waste Matters</b></br>
 
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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!
 
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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>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.
 
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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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<br><p><center><img src="https://static.igem.org/mediawiki/2014/f/f5/Screenshot_from_2014-10-17_18-05-39.png" alt="Smiley face" width="450" height="80"></center></p></br>
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<center>Figure 1. All organisms were grown on M9 Media with specified amounts of Theobromine & Caffeine. Refer to lab notebook.[reduce font size] </center>
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<br>
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By verifiying that our ΔguaB cells could only grow in media with caffeine or theobromine present, we verified that part BBa_K734000 was functional. Subsequent experiments involved monitoring growth through an arrary of theobromine amounts. HPLC results indiciating linear degradation of theobromine have yet to arrive!
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<h2> Reconstruction of
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<h2>Yogurt</h2>
 
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    <p><b>Let's get cultured.</b>
 
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<br>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!
 
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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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Revision as of 23:37, 17 October 2014


Results


"I have had my results for a long time: but I do not yet know how I am to arrive at them."



Karl Friedrich Gauss


Feeding Assay


Experimental analysis began with verifying biobrick BBa_K734000. To verify growth from solely caffeine or theobromine, the organisms were sent to us from UT Austin with the guaB gene knocked out. Guanine comes from xanthine, which is a necessary factor for microbial growth. Without the ability to produce guanine, xanthine degradation is imperative for guanine biosynthesis therefore addicting the microorganisms to caffeine. We set out experimentally to show that theobromine degradation could also be used for xanthine degradation using the same N-Demethylation pathway. Our results are as follow:

Smiley face


Figure 1. All organisms were grown on M9 Media with specified amounts of Theobromine & Caffeine. Refer to lab notebook.[reduce font size]

By verifiying that our ΔguaB cells could only grow in media with caffeine or theobromine present, we verified that part BBa_K734000 was functional. Subsequent experiments involved monitoring growth through an arrary of theobromine amounts. HPLC results indiciating linear degradation of theobromine have yet to arrive!

Reconstruction of


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