Team:HUST-China

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                <a href="https://2013.igem.org/Team:HUST-China/Results">RESULTS</a>
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                <a href="https://2013.igem.org/Team:HUST-China/Protocol">PROTOCOL</a>
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<div class="row" id="abstract" style="width:900px;height:250px;margin:20px 0 30px 50px;border:1px solid #fff;box-shadow:1px 1px 15px 5px rgba(0, 0, 0, 0.5)">
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<div style="float:left;width:240px;text-align:center;margin-top:105px;"><h1>Abstract</h1></div>
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Hypertension has become the leading risk factor for mortality worldwide. Human’s blood pressure (BP) has a basic daily rhythm with two
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                                        peaks,6:00 to 10:00 in the
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morning and 16:00 to18:00 in the afternoon.
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                                        The morning peak is also called “death
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time”,for it’s unlikely to take drugs before waking. Propionate, a short chain fatty acid,
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            <h1 class="page-header">Abstract</h1>
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was recently shown to produce an acute hypotensive response.2013 HUST-China iGEM use a synthetic way to combine bio-oscillator with propionate-producing enzyme
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            <p>Hypertension is sometimes called "<strong>silent killer</strong>", for you don't have any symptoms when actually your blood pressure is far beyond the healthy level, and for it has been identified as a risk factor for coronary artery disease (CAD) and Chronic renal failure (CRF). Although it causes grave concern worldwide for its notoriety, there are not many therapeutic methods to hypertension besides a wide selection among various antihypertensive drugs. However, this comes along with heavy financial burden to the developing countries or underdeveloped countries. In addition, almost all these drugs have side effects to liver and renal.<br>
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gene, trying to build a gut probiotic which can release propionate
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Suppose there is a group of friendly engineering bacteria in the human intestine and they can produce short-chain fatty acids (SCFA) periodically and naturally to help maintain the blood pressure in safe level. Will it be a novel method to treat Hypertension?<br>
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periodically in accord with the rhythm of human BP. This could be a great substitute for chemical drugs by saving patients from drug dependence and the risk of sudden death at
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<strong>SCFA</strong>, especially acetate and propionate, has been proved to induce vasodilatation and ensuing hypotensive response via receptors in smooth muscle cells of vessels. This year, iGEM-HUST have found a metabolic pathway in Escherichia coli (E.coil) that converts succinate to propionate through Wood-Werkman reaction. An operon consisting four genes encodes enzymes in this pathway. By combining bio-oscillator and key gene together, we want to make E. Coli release propionate periodically in patients’ intestine periodically. Once the E.coli is delivered into human body as probiotics, the propionate can be taken by the circulatory system and act with the receptors. However, all the works we have done at present were processed in vitro since we are not sure about the effective concentration for therapy in different patients. And what we are considering is how to prolong the period of propionate and correspond with the peak valley of blood pressure.  
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morning BP peak time.
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            <h1 class="page-header">Overview</h1>
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            <strong class="lead">A four gene operon
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<a href="https://static.igem.org/mediawiki/2013/4/44/HUST-home-figure1.png" target="_blank">Figure 1</a> in Escherichia coli genome which include sbm(scpA) ygfG ygfH ygfD, is significant in the metabolic pathway that converts succinate to propionate through Wood-Werkman reaction. </strong><a href="https://static.igem.org/mediawiki/2013/4/44/HUST-home-figure1.png" target="_blank"><img class="img-polaroid" src="https://static.igem.org/mediawiki/2013/4/44/HUST-home-figure1.png" style="float:right;margin-top:30px;"/></a>
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<span style="font-size:36px;"><a href="https://2013.igem.org/Team:HUST-China/Project">Oscillator</a></span>
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<blockquote>Sbm encodes methylmalonyl-CoA epimerase which catalyzes the reversible reaction of succinyl-CoA and methylmalonyl-CoA, the first step in the propionate synthetic pathway. YgfG encoding by the third gene in the operon, catalyzes the decarboxylation of methylmalonyl-CoA to propionyl-CoA. And YgfH catalyzes a CoA transferase reaction from propionyl-CoA to succinyl, generating propionate. Yet the function of YgfD is not as clear as the remaining three. According to Toomas Haller, the protein encoded by the second gene, YgfD, contains a consensus binding sequence for ATP. They thought it might be a succinate (or propionate) CoA ligase, or a novel (biotin-independent) propionyl-CoA carboxylase. So far, what we confirm is its indispensable importance in the pathway.</blockquote>
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<span><a href="https://2013.igem.org/Team:HUST-China/Project"><img src="https://static.igem.org/mediawiki/2013/3/32/HUST-oscillator.png" style="width:190px;margin-left:10px;"></a></span>
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The key part of the oscillator is araBAD/ lacZYA hybrid p-
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romoter. It is activated by the AraC protein in the prese-
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nce of arabinose and repressed by the LacI protein in the absence of IPTG, constructing two feedback loops with opposite effects.And the differential activity of the two feedback loops can drive oscillatory behaviour.
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            <strong>Reference</strong>
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<div id="propionate" style="float:left;width:408px;height:320px;text-align:left;padding:0 20px;border:1px solid #fff;border-left:none;">
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            <li>Jennifer L. Pluznick, Ryan J. Protzko, Haykanush Gevorgyan, Zita Peterlin, Arnold Sipos, Jinah Han,ect. Olfactory receptor responding to gut microbiota-derived signals plays a role in renin secretion and blood pressure regulation. PNAS Early Edition, Approved January 4, 2013.</li>
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<div>
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            <li>RIVERS SINGLETON, JR. Heterotrophic CO2-Fixation, Mentors, and Students: The Wood-Werkman ReactionS. Journal of the History of Biology 30: 91–120, 1997</li>
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<span style="display:inline-block;font-size:32px;width:190px;margin-top:14px;"><a href="https://2013.igem.org/Team:HUST-China/Project">Propionate<br/><br/>Reactor</a></span>
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            <li>Anne Thierry, Stéphanie-Marie Deutsch, Hélène Falentin, Marion Dalmasso, Fabien J. Cousin, Gwenaël Jan. New insights into physiology and metabolism of Propionibacterium freudenreichii. International Journal of Food Microbiology 149 (2011) 19 – 27</li>
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<span><a href=""><a href="https://2013.igem.org/Team:HUST-China/Project"><img src="https://static.igem.org/mediawiki/2013/9/9a/HUST-hplc.jpg" style="width:190px;margin-left:10px;"></a></span>
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            <li>Toomas Haller, Thomas Buckel, Ja ´nos Re´tey, and John A. Gerlt. Discovering New Enzymes and Metabolic Pathways: Conversion of Succinate to Propionate by Escherichia coli. Biochemistry2000, 39, 4622-4629</li>
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A four-gene operon in E.coli K12 genome which includes sbm,ygfG,ygfH and ygfD, is significant in the metabolic pathway that converts succinate to propionate through Wood-Werkman reaction. We constructed effective expression plasmid to increase the quantity of the four enzymes independently. By measuring the propionate amount, we figured out which of the four is the most effective.
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<span style="font-size:29px;"><a href="https://2013.igem.org/Team:HUST-China/Project">Future work</a></span>
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In the near future, we will regulate the period of propionate utilizing the frequency divider with a ssrA-tag analog attached to the end of enzyme.Besides,we will replace mRFP with key gene in the synthetic pathway, hoping to see periodical release of propionate in accord with the rhythm of human BP.
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<span style="display:inline-block;font-size:37px;width:190px;margin-top:14px;"><a href="https://2013.igem.org/Team:HUST-China/Protocol">Protocol</a></span>
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<span><a href="https://2013.igem.org/Team:HUST-China/Protocol"><img src="https://static.igem.org/mediawiki/2013/e/ee/HUST-protocol.jpg" style="width:190px;margin-left:10px;"></a></span>
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Our project is divided into three parts: the construction of the biological oscillator, the output evaluation of propionate by HPLC and the standardization of four genes.
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<span style="font-size:35px;"><a href="https://2013.igem.org/Team:HUST-China/Modelling">Modeling</a></span>
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<span><a href="https://2013.igem.org/Team:HUST-China/Modelling"><img src="https://static.igem.org/mediawiki/2013/e/ee/HUST-modelling.png" style="width:190px;margin-left:10px;"></a></span>
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Detailed analysis of the oscillator makes us clearer about how it works. From the establishment of <acronym title="Delayed Differential Equations">DDEs</acronym> to parameter sweep and sensitivity analysis, we know how each parameter contributes to the period. The <acronym title="Multi Cells Oscillation Simulation">MCOS</acronym> shows us the feasibility of a group of oscillators that can be eventually applied in practice in vivo (colon).Lastly, we statistically analyzed our simulated data and wet-lab data.
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<span style="display:inline-block;font-size:35px;width:190px;margin-top:14px;"><a href="https://2013.igem.org/Team:HUST-China/HumanPractice">Human <br><br>Practice</a></span>
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<span><a href="https://2013.igem.org/Team:HUST-China/HumanPractice"><img src="https://static.igem.org/mediawiki/2013/1/11/HUST-humanpractice.jpg" style="width:190px;margin-left:10px;"></a></span>
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We have done a remarkable job in introducing high school students synthetic biology and
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iGEM jamborees as well as motivating them towards future participation in the iGEM. Besides, we collaborated with two other iGEM teams by sharing plasmids and characterizing their parts. Furthermore, we made  public speeches in our school about what we achieved,shared the felt in the iGEM competition.
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                 <div class="modal-footer footer"><h2>Acknowledgement</h2><hr>
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<strong>Acknowledgement</strong>
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<a href="http://life.hust.edu.cn" target="_blank" title="Life Science and Technology of HUST"><img src="https://static.igem.org/mediawiki/2013/a/a5/HUST-life.png" width="100px" height="100px"/></a>
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<a href="http://english.hust.edu.cn" target="_blank" title="Huazhong University of Science and Technology"><img src="https://static.igem.org/mediawiki/2013/0/00/HUST-hust.jpg" width="120px" height="100px"/></a>
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</span>
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<span style="display:inline-block;float:left;margin:0 20px 20px 0;">
<a href="http://qiming.hust.edu.cn" target="_blank" title="Qiming College of Huazhong University of Science and Technology"><img src="https://static.igem.org/mediawiki/2013/4/46/HUST-qiming.png" /></a>
<a href="http://qiming.hust.edu.cn" target="_blank" title="Qiming College of Huazhong University of Science and Technology"><img src="https://static.igem.org/mediawiki/2013/4/46/HUST-qiming.png" /></a>
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CopyRight &copy;<a href="https://2013.igem.org">2013.igem.org</a>&nbsp;All Rights Reserved!
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<div>Powered by the iGEM team comes from HuaZhong University of Science and Techology&nbsp;
<div>Powered by the iGEM team comes from HuaZhong University of Science and Techology&nbsp;
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<a href="http://www.hust.edu.cn" target="_blank" title="HUST-China">HUST-China</a>
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<a href="http://english.hust.edu.cn" target="_blank" title="HUST-China">HUST-China</a>
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Latest revision as of 10:03, 28 October 2013

Abstract

Hypertension has become the leading risk factor for mortality worldwide. Human’s blood pressure (BP) has a basic daily rhythm with two peaks,6:00 to 10:00 in the morning and 16:00 to18:00 in the afternoon. The morning peak is also called “death time”,for it’s unlikely to take drugs before waking. Propionate, a short chain fatty acid, was recently shown to produce an acute hypotensive response.2013 HUST-China iGEM use a synthetic way to combine bio-oscillator with propionate-producing enzyme gene, trying to build a gut probiotic which can release propionate periodically in accord with the rhythm of human BP. This could be a great substitute for chemical drugs by saving patients from drug dependence and the risk of sudden death at morning BP peak time.
The key part of the oscillator is araBAD/ lacZYA hybrid p- romoter. It is activated by the AraC protein in the prese- nce of arabinose and repressed by the LacI protein in the absence of IPTG, constructing two feedback loops with opposite effects.And the differential activity of the two feedback loops can drive oscillatory behaviour.
A four-gene operon in E.coli K12 genome which includes sbm,ygfG,ygfH and ygfD, is significant in the metabolic pathway that converts succinate to propionate through Wood-Werkman reaction. We constructed effective expression plasmid to increase the quantity of the four enzymes independently. By measuring the propionate amount, we figured out which of the four is the most effective.
In the near future, we will regulate the period of propionate utilizing the frequency divider with a ssrA-tag analog attached to the end of enzyme.Besides,we will replace mRFP with key gene in the synthetic pathway, hoping to see periodical release of propionate in accord with the rhythm of human BP.
Our project is divided into three parts: the construction of the biological oscillator, the output evaluation of propionate by HPLC and the standardization of four genes.
Detailed analysis of the oscillator makes us clearer about how it works. From the establishment of DDEs to parameter sweep and sensitivity analysis, we know how each parameter contributes to the period. The MCOS shows us the feasibility of a group of oscillators that can be eventually applied in practice in vivo (colon).Lastly, we statistically analyzed our simulated data and wet-lab data.
We have done a remarkable job in introducing high school students synthetic biology and iGEM jamborees as well as motivating them towards future participation in the iGEM. Besides, we collaborated with two other iGEM teams by sharing plasmids and characterizing their parts. Furthermore, we made public speeches in our school about what we achieved,shared the felt in the iGEM competition.

Acknowledgement


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