Team:UIUC Illinois/Project/PuppyProbiotic

From 2014.igem.org

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<center><p style="font-size:60px">Puppy Probiotics.</p></center>
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<center><p style="font-size:30px"><i>"The art of research [is] the art of making difficult problems soluble by devising means of getting at them. "</i></p></center>
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<br><center><p style="font-size:15px">- Sir Peter Medawar</p></br></center>
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<center><h2>An Introduction</h2></centeR>
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<br><p style="font-size:15px">Theobromine is a 3,7- dimethylxanthine which has a bitter taste and is naturally present in many substances as well as a metabolite of many chemical cascades. Theo being the greek root for “god” and broma meaning “food” combined form Theobroma, the genus of the cacao tree which is used in chocolate formation. Despite its abundant and delicious presence in chocolates and tea leaves, Theobromine poses a great risk to animals who are not made to digest it. Dogs who are exposed to chocolate and subsequently theobromine exhibit high blood pressure, can begin to seize and as many veterinary clinics have reported, result in death. To combat this issue, the UIUC iGEM team is focused on creating a probiotic that when administered, can combat the ill effects of theobromine on livestock and domesticated animals. We explore the potential benefits of the ever increasing manufacture of probiotics as well as colonization of digestive tracks to supress negative chemical effects.
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  <h2> Puppy Probiotics</h2>
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In our venture to degrade theobromine, we utilized cascades that broke down not only theobromine, but its precursor caffeine. Caffeine and theobromine are both nervous stimulants and vasodilators which can be beneficial or malignant depending on context. Examining the scope of coffee production, we discovered that despite being a rich source of nitrogen and carbon, coffee waste is not as widely used as biomass for fertilizers due to its caffeine content. We aimed to provide a way to remediate coffee waste to make it a viable feed source.</p>
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  <p><b> Safety forms were approved on Date, 2014 by John Doe.</b></p>
 
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  <h2>Personal Safety of Members in the Lab</h2>
 
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    <p><b>Biosafety Training:</b>
 
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      All team members were required to attend safety training focused on (1)Understanding what bloodborne pathogens are and their potential impact on your health,
 
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(2)Learning how to best protect yourself from exposure to bloodborne pathogens, and (3)Knowing what to do if you are exposed to potentially infectious material.
 
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    <p><b>Known toxic chemical reagents and hazardous physical agents:</b>
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<br><b><p style="font-size:15px">Imagine...</p></b></br>
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Ethidium Bromide: Since ethidium bromide is a very toxic agent.  
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  <p style="font-size:15px">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.</p>
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We have separate containers for tips, gels and pipettes that have the possibility to carry ethidium bromide.  
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Gloves used in that area are changed immediately after the experiments there.<br>
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Ultraviolet Light: UV radiation is used for visualization of the stained DNA in the electrophoretic gels.
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We always use UV-blocking shields when visualizing electrophoretic gels in UV light.
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All other toxic and chemicals will be handled to avoid direct contact, and observing the proper safety procedures, additionally,
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all chemicals reagents and biological materials will be disposed only in the designated biohazard receptacles.
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     <p><b>Personal Safety</b>
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     <p><b><p style="font-size:15px">What just happened?</p></b>
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<br><p style="font-size:15px">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!</p>
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<li>Laboratory coveralls, appropriate gloves, safety googles are worn. After use, gloves should be removed aseptically and hands will then be washed.</li>
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<br><center><img src="https://static.igem.org/mediawiki/2014/7/7a/Theobromine-molecule.gif" alt="Smiley face" width="350" height="350"></center></br>
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<li>Personnel must wash their hands after handling infectious materials and animals, and before they leave the laboratory working areas.</li>
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<li>Safety glasses, face shields (visors) or other protective devices must be worn when it is necessary to protect the eyes and face from splashes, impacting objects and sources of artificial ultraviolet radiation.</li>
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<li>It is prohibited to wear protective laboratory clothing outside the laboratory, e.g. in canteens, coffee rooms, offices, libraries, staff rooms and toilets.</li>
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<li>Open-toed footwear must not be worn in laboratories.</li>
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<li>Eating, drinking, smoking, applying cosmetics and handling contact lenses is prohibited in the laboratory working areas.</li>
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<li>Storing human foods or drinks anywhere in the laboratory working areas is prohibited.</li>
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<li>Protective laboratory clothing that has been used in the laboratory must not be stored in the same lockers or cupboards as street clothing.</li>
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   <h2> Training the Team Underwent </h2>
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   <h2> What can we do? </h2>
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     <p><b>Have your team members received any safety training yet?</b>
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     <p><b><p style="font-size:15px">Problem solving</p></b>
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<br><p style="font-size:15px">In effort to solve this problem, our team looked at the biological cascade of caffeine and theobromine. Since our goal was to degrade caffeine into something less harmful, we looked to digestion. We uncovered that theobromine can in fact be degraded into subtituents that are less harmful, namely a cascade that converts it to uric acid, a molecule that the dogs could excrete as waste</p></br>
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      Our team had two types of training. The first was an online training by the DRS or the Division of Research Safety. This safety training included multiple quizzes and tutorials for General Laboratory Safety and Understanding BioSafety. Additionally, our team took a synthetic biology bootcamp at the beginning of the year to cover general laboratory procedures and safety guidelines. Other safety training such as autoclaving came at another time.</p>
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<img src="https://static.igem.org/mediawiki/2014/1/1b/Picture1.png" alt="Smiley face" width="500" height="650">
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    <p><b>Please briefly describe the topics that you learned about (or will learn about) in your safety training.</b>
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      For all techniques listed, our lab required usage of gloves, goggles, lab coats, closed-toe shoes and long pants to minimize the amount of contact between us and the materials that are being used. Also, general aseptic protocols were adhered to when handling any lab techniques to prevent contamination. Our lab required us to go through a live training with a lab representative to learn proper handling and care of the autoclave.
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    <p><b>Who is responsible for biological safety at your institution? </b>
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<br><b><p style="font-size:15px">Nature's answer</p></b></br>
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      BioSafety is all handled by the Division of Research Safety or DRS. As long as it is BioSafety Level 1, our lab, The Institute of Genomic Biology, has no concerns and just requires us to take training through the DRS. The training for the DRS includes online presentations to teach us safety techniques. These presentations are followed by online quizzes that test what we have learn to earn certification. The certification enables us usage of the lab.  
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    <p style="font-size:15px">We discovered that the organism Pseudomonas putida CBB5 endogenously metabolized caffeine and subsequent methylxanthines. In fact, the organism degraded the compounds using the proteins NdmA (N-Demethylase A) and NdmB, both of which are Rieske nonheme iron monooxygenases, NdmC, another demethylase and NdmD, a reductase. By using this pathway, the organism is able to use caffeine as a carbon and nitrogen source.[2] Too good to be true? No. In fact, a previous iGEM team, UT Austin 2012 Caffeinated Coli, harnessed the power of this novel pathway in their own project! We were ecstatic to learn that the pathway was available in biobrick form and ready for us to manipulate.</p>
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<img src="https://static.igem.org/mediawiki/2012/f/fc/Ndm_diagram.JPG" alt="Smiley face" width="500" height="200">
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<br><p style="font-size:15px">UT Austin developed this part by rearranging the operon of CBB5 and developed this part:</p>
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<img src="https://static.igem.org/mediawiki/2012/2/25/Austin_Texas_Decaffeination_design.png" alt="Smiley face" width="500" height="100">
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<br><p style="font-size:15px">By using this construct, we were eager to repurpose it to degrade theobromine. Degrading theobromine meant targeting the host where theobromine failed to be degraded. An enteric approach is what we chose, thus leading us to design a probiotic. We chose Latobacillus, specifically Lactobacillus Plantarum WCFS1 as our fermenter of choice. Our plan was to PCR out the genes corresponding to theobromine degradation (NdmB, NdmC, NdmD) and assemble that into a shuttle vector for Lactobacillus.</p>
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<br><b><p style="font-size:15px">Plantarum to propagate the Puppy Probiotic</p></b></br>
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<p style="font-size:15px">How could we deliver our theobromine degrading enzymes to the gut of the host? We decided upon the ever increasingly popular probiotic. Probiotics are foods or supplements that harness microbes to produce products for a positive purpose. To create a probiotic, delivery of an organism was necessary. Lactobacillus Plantarum WCFS1 is a strain of Lactobacillus that is capable of surviving gut conditions as well as thriving in the endogenous microbiota of the organism it's in. By having Plantarum in the gut, we hoped that it would produce enzymes that could survive and propagate in the gut itself. Plantarum became the organism we decided to transform our products into.</p>
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<center><img src="https://static.igem.org/mediawiki/2014/e/e6/Plantarumprobiotic.jpg" alt="Smiley face" width="200" height="300"></center>
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Latest revision as of 03:20, 18 October 2014


Puppy Probiotics.

"The art of research [is] the art of making difficult problems soluble by devising means of getting at them. "


- Sir Peter Medawar


An Introduction


Theobromine is a 3,7- dimethylxanthine which has a bitter taste and is naturally present in many substances as well as a metabolite of many chemical cascades. Theo being the greek root for “god” and broma meaning “food” combined form Theobroma, the genus of the cacao tree which is used in chocolate formation. Despite its abundant and delicious presence in chocolates and tea leaves, Theobromine poses a great risk to animals who are not made to digest it. Dogs who are exposed to chocolate and subsequently theobromine exhibit high blood pressure, can begin to seize and as many veterinary clinics have reported, result in death. To combat this issue, the UIUC iGEM team is focused on creating a probiotic that when administered, can combat the ill effects of theobromine on livestock and domesticated animals. We explore the potential benefits of the ever increasing manufacture of probiotics as well as colonization of digestive tracks to supress negative chemical effects. In our venture to degrade theobromine, we utilized cascades that broke down not only theobromine, but its precursor caffeine. Caffeine and theobromine are both nervous stimulants and vasodilators which can be beneficial or malignant depending on context. Examining the scope of coffee production, we discovered that despite being a rich source of nitrogen and carbon, coffee waste is not as widely used as biomass for fertilizers due to its caffeine content. We aimed to provide a way to remediate coffee waste to make it a viable feed source.



Imagine...


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.

Smiley face

What just happened?


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!


Smiley face

What can we do?

Problem solving


In effort to solve this problem, our team looked at the biological cascade of caffeine and theobromine. Since our goal was to degrade caffeine into something less harmful, we looked to digestion. We uncovered that theobromine can in fact be degraded into subtituents that are less harmful, namely a cascade that converts it to uric acid, a molecule that the dogs could excrete as waste


Smiley face


Nature's answer


We discovered that the organism Pseudomonas putida CBB5 endogenously metabolized caffeine and subsequent methylxanthines. In fact, the organism degraded the compounds using the proteins NdmA (N-Demethylase A) and NdmB, both of which are Rieske nonheme iron monooxygenases, NdmC, another demethylase and NdmD, a reductase. By using this pathway, the organism is able to use caffeine as a carbon and nitrogen source.[2] Too good to be true? No. In fact, a previous iGEM team, UT Austin 2012 Caffeinated Coli, harnessed the power of this novel pathway in their own project! We were ecstatic to learn that the pathway was available in biobrick form and ready for us to manipulate.

Smiley face

UT Austin developed this part by rearranging the operon of CBB5 and developed this part:

Smiley face

By using this construct, we were eager to repurpose it to degrade theobromine. Degrading theobromine meant targeting the host where theobromine failed to be degraded. An enteric approach is what we chose, thus leading us to design a probiotic. We chose Latobacillus, specifically Lactobacillus Plantarum WCFS1 as our fermenter of choice. Our plan was to PCR out the genes corresponding to theobromine degradation (NdmB, NdmC, NdmD) and assemble that into a shuttle vector for Lactobacillus.






Plantarum to propagate the Puppy Probiotic


How could we deliver our theobromine degrading enzymes to the gut of the host? We decided upon the ever increasingly popular probiotic. Probiotics are foods or supplements that harness microbes to produce products for a positive purpose. To create a probiotic, delivery of an organism was necessary. Lactobacillus Plantarum WCFS1 is a strain of Lactobacillus that is capable of surviving gut conditions as well as thriving in the endogenous microbiota of the organism it's in. By having Plantarum in the gut, we hoped that it would produce enzymes that could survive and propagate in the gut itself. Plantarum became the organism we decided to transform our products into.

Smiley face


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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