Team:HUST-China

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<div style="float:left;width:240px;text-align:center;margin-top:105px;"><h1>Abstract</h1></div>
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          <li class="active"><a href="#abstract"><i class="icon-chevron-right"></i>Abstract</a></li>
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          <li><a href="#overview"><i class="icon-chevron-right"></i>Overview</a></li>
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Hypertension has become the leading risk factor for mortality worldwide. Human’s blood pressure has a basic daily rhythm with two peaks,
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6:00 to 10:00 in the morn-
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ing and 16:00 to18:00 in the afternoon.The
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morning peak is also called “death tim-
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e”,for it’s unlikely to take drugs before waking. Propionate, a short chain fatty acid,
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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 enzy-
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            <h1 class="page-header">Abstract</h1>
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me gene, trying to build a gut probiotic which can release pro-
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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. Patients have to take those drugs throughout rest of their lives once diagnosed with hypertension. 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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pionate 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 m-
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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 a safe range. Will it be a novel method to treat Hypertension?<br>
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orning BP peak time.
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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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            <h1 class="page-header">Overview</h1>
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            <strong class="lead">A four gene operon
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<span style="font-size:40px;">Oscillator</span>
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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><a href=""><img src="./images/oscillator.png" style="width:190px;margin-left:10px;"></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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The key part of the oscillator is araBAD/ lacZYA hybrid promoter. It is activated by the AraC protein in the pre-
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sence of arabinose and repressed by the LacI protein in the absence of IPTG. Hence, activation of the promoters by the addition of Arabinose and IPTG to the medium r-
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esults in transcription of each component of the circuit.
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            <strong>Reference</strong>
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Increased production of AraC in the presence of arabin-
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ose results in a positive feedback loop that increases p-
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romoter activity. However, the concurrent increase in production of LacI results in a linked negative feedback loop that decreases promoter activity. The differential activity of the two feedback loops can drive oscillatory behavior.
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            <li><a style="font-weight:normal;" href="https://static.igem.org/mediawiki/2013/7/76/HUST-1.pdf" target="_blank">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.</a></li>
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            <li><a style="font-weight:normal;" href="https://static.igem.org/mediawiki/2013/3/36/HUST-2.pdf" target="_blank">RIVERS SINGLETON, JR. Heterotrophic CO2-Fixation, Mentors, and Students: The Wood-Werkman ReactionS. Journal of the History of Biology 30: 91–120, 1997</a></li>
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            <li><a style="font-weight:normal;" href="https://static.igem.org/mediawiki/2013/6/6a/HUST-3.pdf" target="_blank">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</a></li>
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            <li><a style="font-weight:normal;" href="https://static.igem.org/mediawiki/2013/c/c1/HUST-4.pdf" target="_blank">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</a></li>
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<span style="display:inline-block;font-size:37px;width:190px;margin-top:14px;">Propionate<br/><br/>reactor</span>
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<span><a href=""><img src="./images/hplc.jpg" style="width:190px;margin-left:10px;"></a></span>
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A four gene operon in E.coli K12 genome which includes sbm ygfG ygfH ygfD, is significant in the metabolic pathway that converts succinate to propionate through Wood-Werkman reaction. We construct effective expression plasmid to increase the quantity of the four enzymes independently. By detecting the propionate amount by HPLC, we get to know which of the four is the rate-limiting enzyme. We choose it as the output of the oscillator.
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<span style="font-size:33px;">Future work</span>
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In the future, we will use the gene of rate-limiting enzyme of propionate producing reaction to replace mRFP in the oscillator as the output. And we hope to see periodical r-
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elease of propionate.
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Later ,we may use mouse model to demonstrate our original design.
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<div style="float:left;width:408px;height:255px;text-align:left;padding:0 20px;border:1px solid #fff;border-left:none;">
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<span style="display:inline-block;font-size:37px;width:190px;margin-top:14px;">Protocal</span>
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<span><img src="./images/protocol.jpg" style="width:190px;margin-left:10px;"></span>
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Aside of traditional molecular biology method, we mainly use HPLC to detect propionate, flow cytometer(FCM) and confocal to detect fluorescence.
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<span style="font-size:37px;">Modeling</span>
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<span><img src="./images/modelling.png" style="width:190px;margin-left:10px;"></span>
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Detailed analysis of the oscillator makes us clearer about how it works. From the establishment of DDEs (delayed differential equations) to parameter sweep and sensitivity analysis, we know how each parameter contributes to the period. The MCOS (MultiCell Oscillation Simulation)shows us the exact feasibility of the oscillator both in vitro and in vivo (colon).
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<span style="display:inline-block;font-size:37px;width:190px;margin-top:14px;">Human <br><br>practice</span>
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<span><img src="./images/humanpractice.jpg" style="width:190px;margin-left:10px;"></span>
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Human practice:We have done a remarkable job in intro-
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ducing high school students to synthetic biology and
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iGEM jamborees as well as motivating them towards fu-
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ture participation in the iGEM. Besides, we collaborated with two other iGEM teams by sharing DNA materials and characterizing their parts. Furthermore, we made a public speech in our school to report what we achieved, to sha-
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re the feelings in the iGEM competition.And we also desi-
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gn a crossword puzzle about iGEM for fun of all iGEMers.
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Revision as of 06:19, 27 September 2013

Abstract

Hypertension has become the leading risk factor for mortality worldwide. Human’s blood pressure has a basic daily rhythm with two peaks, 6:00 to 10:00 in the morn- ing and 16:00 to18:00 in the afternoon.The morning peak is also called “death tim- e”,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 enzy- me gene, trying to build a gut probiotic which can release pro- pionate 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 m- orning BP peak time.
Oscillator
The key part of the oscillator is araBAD/ lacZYA hybrid promoter. It is activated by the AraC protein in the pre- sence of arabinose and repressed by the LacI protein in the absence of IPTG. Hence, activation of the promoters by the addition of Arabinose and IPTG to the medium r- esults in transcription of each component of the circuit. Increased production of AraC in the presence of arabin- ose results in a positive feedback loop that increases p- romoter activity. However, the concurrent increase in production of LacI results in a linked negative feedback loop that decreases promoter activity. The differential activity of the two feedback loops can drive oscillatory behavior.
Propionate

reactor
A four gene operon in E.coli K12 genome which includes sbm ygfG ygfH ygfD, is significant in the metabolic pathway that converts succinate to propionate through Wood-Werkman reaction. We construct effective expression plasmid to increase the quantity of the four enzymes independently. By detecting the propionate amount by HPLC, we get to know which of the four is the rate-limiting enzyme. We choose it as the output of the oscillator.
Future work
In the future, we will use the gene of rate-limiting enzyme of propionate producing reaction to replace mRFP in the oscillator as the output. And we hope to see periodical r- elease of propionate. Later ,we may use mouse model to demonstrate our original design.
Protocal
Aside of traditional molecular biology method, we mainly use HPLC to detect propionate, flow cytometer(FCM) and confocal to detect fluorescence.
Modeling
Detailed analysis of the oscillator makes us clearer about how it works. From the establishment of DDEs (delayed differential equations) to parameter sweep and sensitivity analysis, we know how each parameter contributes to the period. The MCOS (MultiCell Oscillation Simulation)shows us the exact feasibility of the oscillator both in vitro and in vivo (colon).
Human

practice
Human practice:We have done a remarkable job in intro- ducing high school students to synthetic biology and iGEM jamborees as well as motivating them towards fu- ture participation in the iGEM. Besides, we collaborated with two other iGEM teams by sharing DNA materials and characterizing their parts. Furthermore, we made a public speech in our school to report what we achieved, to sha- re the feelings in the iGEM competition.And we also desi- gn a crossword puzzle about iGEM for fun of all iGEMers.
Acknowledgement

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