Team:HUST-China/Project

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                <a href="https://2013.igem.org/Team:HUST-China/Results">RESULTS</a>
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             <h1 class="page-header">Abstract</h1>
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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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             <h1 class="page-header"><strong>Overview</strong></h1>                                      
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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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            <p><b><font size="4px">Hypertension</font></b>is a worldwide public health challenge. And it has been identified as the leading risk factor for mortality mainly because it can lead to adverse cardiovascular events. These events appear to follow a circadian patter, reaching a peak in the morning shortly after wakening and arising. Why? That’s because human’s blood pressure (BP) follows a basic daily rhythm, reaching a peak in the morning when you wake up, then going down and reversing up at 4:00 in the afternoon. (As show in fig.1.1) It is likely that a patient dead in the dream even he felt nothing wrong in the daytime but actually the blood pressure is far beyond the healthy level in the daybreak. Traditional drugs have difficulty to solve the problem as
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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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It’s not possible to take pills when asleep.
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            <strong class="lead">A four gene operon Figure1 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><img class="img-polaroid" src="https://static.igem.org/mediawiki/2013/4/44/HUST-home-figure1.png" style="float:right;margin-top:30px;"/>
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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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<p><em style="font-size:11px;">Figure1.1: human blood pressure daily rhythm</em></p>
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<p>To cope with the situation, our team HUST-China tries to construct a group of friendly probiotic bacteria with the ability of releasing hypotensor in step with human BP daily rhythm in patients’ intestine. In that case, patients will not worry about excessive morning surge anymore. This can be a novel approach to hypertension.</p>
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<p>What can act as the hypotensor? The answer is short chain fatty acid (SCFA).
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SCFA, especially propionate was newly proved to cause an acute hypotensive response by Jennifer et al. A GPCRs called olfr78 expressed in smooth muscle cells of small blood vessel plays an important role. It could be activated by propionate and induce vasodilatation and hypotension.
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<p>The question then is: how can we make the hypotensor released periodically?Our solution is bio-oscillator!</p>
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<p>To intervene the bacterium’s generation of propionate. We firstly find a metabolic pathway in E.coil that converts succinate to propionate. (The mechanism of propionate generation is pictured in fig.1.2)There are four enzymes functioning in the pathway. We aimed to find the key gene to be the output of our bio-oscillator.To do this, we recombine the four genes to expression vector, transform them independently and use the four recombinant strains get to do fermentation .Measure propionate concentration by HPLC and find out which gene affects mostly.</p>
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<p><em style="font-size:11px;">Fig1.2: Mechanism of propionate generation</em></p>
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<p>To control the bacterium’s generation of propionate. We use a bio-oscillator. (The design is shown in figure1.3) We firstly use an mRFP as reporter to test and verify our design.</p>
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<p><em style="font-size:11px;"> Figure1.3: Design of our bio-oscillator</em></p>
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<p>After finishing these two main works,we will replace mRFP with our key gene.Regulating the period of oscillator utilizing the frequency divider with an ssrA-tag analog attached to the end of enzyme.<br>
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  We believe our oscillating BP reliever is both helpful and practical.
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            <h1 class="page-header"><strong>Results</strong></h1>
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                                        <p>As we divided our project to two main parts: propionate generation and oscillator. The results will be demonstrated from these two sections.</p>
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<h4>Propionate generation:</h4>
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            <p>1.We firstly successfully copied the four genes ygfD/ygfH/ygfG/Sbm from E.coli strain K12, and then recombined them independently to expression vector.  Purified protein from cell disruptions by Ni-chelating and did gel analysis. From figure-1 , we confirmed that the enzymes were successfully expressed.</p>
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<div style="text-align:center;"><p><img src="https://static.igem.org/mediawiki/2013/6/6e/HUST-proj-res1.png" /></p>
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<p><em  style="font-size:11px;">Figure1. SDS-PAGE analysis of Sbm, ygfD, ygfG, ygfH over-expression</em></p>
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<p>2.As we thought the overexpression of enzymes didn’t directly prove the increase of propionate, and we wanted to find the key gene in the reaction. So we used the positive clones to do fermentation. We firstly optimized the condition and found the best sampling time points. Then we used standard propionate of seven gradients to draw a linear graph between propionate concentration and HPLC peak area. Figure-2 shows the relationship.</p>
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<div style="text-align:center;"><p><img src="https://static.igem.org/mediawiki/2013/7/76/HUST-proj-res2.png" /></p>
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<p><em  style="font-size:11px;">Figure-2. Standard curve of propionate and HPLC peak area</em></p>
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<p>Later, we used the recombinant strain to do fermentation, and measured propionate concentration in the samples. Figure-3 shows the propionate increase percent of each gene recombinant strain. From the data, we found propionate production had a significant increase when ygfD transformed. In other words, we found the key gene- our oscillator’s output.</p>
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<div style="text-align:center;"><p><img src="https://static.igem.org/mediawiki/2013/0/05/HUST-proj-res3.png" /></p>
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<p><em  style="font-size:11px;">Figure-3. HPLC analysis wild-type BL21 and recombination BL21 with four genes</em></p>
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<h4>Oscillator:</h4>
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<p>1.As for the oscillator, we first successfully constructed the two plasmids formed the dual-feedback circuit. (Circuit and plasmid were show in figure-4) LAA tag was added to the protein for rapid degradation. Gene sequencing confirmed the plasmids to be correct without any lethal mutation.</p>
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<div style="text-align:center;"><p><img src="https://static.igem.org/mediawiki/2013/0/08/HUST-proj-res4.png" /></p>
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<p>Figure-4.The dual-feedback circuit and two plasmids we constructed</p>
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<p>2.After that, we transformed recombinant plasmid pET-28a (+) which had an mRFP reporter to check if it could function well. We induced the positive clone with 2mM IPTG and observed by fluorescence microscope. Figure-5 shows that it function well.</p>
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<div style="text-align:center;"><p><img src="https://static.igem.org/mediawiki/2013/8/83/HUST-proj-res5.png" /></p>
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<p><em  style="font-size:11px;">Figure-5. Fluorescence microscope photo of recombinant plasmid pET-28a (+) transformed cell induced by 2mM IPTG</em></p>
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<p>3.Later, we co-transformed two recombinant plasmids pET-28a (+) and pACYCDuet-1. After inducing positive clone by 0.7% Arabinose and 2 mM IPTG, we used fluorescence microscope to see RFP change of single cell and used fluorospectro photometer to see RFP change of multicells.Figure-6 & Figure-7 shows the result.</p>
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<div style="text-align:center;"><p><img src="https://static.igem.org/mediawiki/2013/e/ec/HUST-proj-res6.png" /></p>
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<p><em  style="font-size:11px;">Figure-6. Fluorescence change of single cell</em></p>
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<p>Cells were diluted with culture medium and immobilized with glycerol. (5ul bacteria +20ulmedium+20ul glycerol). Making sure that the cell was alive and motionless, we could take photo of the same cell. From the pictures, we can see that the fluorescence changed with time in an oscillatory way, which supports our bio-oscillator design.</p>
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<div style="text-align:center;"><p><img src="https://static.igem.org/mediawiki/2013/0/01/HUST-proj-res7.jpg" /></p>
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<p><em  style="font-size:11px;">Figure-7. Fluorescence change of multicell</em></p>
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<p>We used fluorospectro photometer to measure the oscillating behavior of multi-cells.
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After induced by 0.7% arabinose and 2 mM IPTG when OD600 was 0.55, cells were cultured in 37℃/200rpm.We sampled a series of time points and draw the curve as figure-7. We saw a significant fluorescence oscillating in compare to control group. The control group cells transformed pET-28a (+) only.</p>
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<p>Summarization: We have successfully found a way to enhance cells generation of propionate, tested and verified our design of bio-oscillator. Combining these two works, we believe we can build a gut probiotic which can release propionate periodically in accord with the rhythm of human BP.</p>
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            <strong>Reference</strong>
 
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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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            <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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            <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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            <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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<strong>Acknowledgement</strong>
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            <div id="Future-Work">
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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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            <h1 class="page-header"><strong>Future work</strong></h1>
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            <p>We do not have enough time to fulfill the whole project. Based on the pre-existing work, these tasks are coming to be finished in future.<br>
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1.Replace the report gene mRFP by ygfD in the dual-feedback circuit to see whether the propionate generation can oscillate.<br>
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<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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2.Regulate the period of propionate generation to cope with human’s BP rhythm. We may utilize the frequency divider with an ssrA-tag analog attached to the end of enzyme to achieve it.<br>
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<div style="float:right"><img src="https://static.igem.org/mediawiki/2013/d/d6/HUST-proj-future-work.png" width="200" /></div>
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3.Transform the regulatory net into bifidobacterium – microorganism has reputation among the dairy industry — due to people who we sent questionnaire to show preference eating food containing probiotics rather than E.coli, Also, we will measure the propionate outside of the human body.<br>
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            <h1 class="page-header"><strong>Judging Critieria</strong></h1>
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            <p>Already registered in the official website in 13th March and was accepted in 12th April. <br>
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1.We completed safety form, judging form and team wiki before the deadline. It is for sure that we are going to present a poster and a talk at the iGEM Jamboree. <br>
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2.We documented four newly standard BioBrick Part(sbm/ygfG/ygfH/ygfD) used in our project and submitted them to the iGEM Registry adhere to guidelines. <br>
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3.Our works aims at maintaining the blood pressure through microbe metabolism SCFA, which is a new application in medicine to our knowledge.<br>
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4.We did plenty of experiment to validate that two of BioBrick Part of our own design and construction works as expected. <br>
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5.We share information and material with WHU and HZAU .Cooperating with HZAU on characterizing one part.<br>
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        6.We originaly creat a crossword to popularize historical knowledge about iGEM. That's a good new approach for human practice.<br>
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        Therefore, we believe that we deserve a Gold Medal Prize.<br>
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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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Latest revision as of 13:58, 28 October 2013

Overview

Hypertensionis a worldwide public health challenge. And it has been identified as the leading risk factor for mortality mainly because it can lead to adverse cardiovascular events. These events appear to follow a circadian patter, reaching a peak in the morning shortly after wakening and arising. Why? That’s because human’s blood pressure (BP) follows a basic daily rhythm, reaching a peak in the morning when you wake up, then going down and reversing up at 4:00 in the afternoon. (As show in fig.1.1) It is likely that a patient dead in the dream even he felt nothing wrong in the daytime but actually the blood pressure is far beyond the healthy level in the daybreak. Traditional drugs have difficulty to solve the problem as It’s not possible to take pills when asleep.

Figure1.1: human blood pressure daily rhythm

To cope with the situation, our team HUST-China tries to construct a group of friendly probiotic bacteria with the ability of releasing hypotensor in step with human BP daily rhythm in patients’ intestine. In that case, patients will not worry about excessive morning surge anymore. This can be a novel approach to hypertension.

What can act as the hypotensor? The answer is short chain fatty acid (SCFA). SCFA, especially propionate was newly proved to cause an acute hypotensive response by Jennifer et al. A GPCRs called olfr78 expressed in smooth muscle cells of small blood vessel plays an important role. It could be activated by propionate and induce vasodilatation and hypotension.

The question then is: how can we make the hypotensor released periodically?Our solution is bio-oscillator!

To intervene the bacterium’s generation of propionate. We firstly find a metabolic pathway in E.coil that converts succinate to propionate. (The mechanism of propionate generation is pictured in fig.1.2)There are four enzymes functioning in the pathway. We aimed to find the key gene to be the output of our bio-oscillator.To do this, we recombine the four genes to expression vector, transform them independently and use the four recombinant strains get to do fermentation .Measure propionate concentration by HPLC and find out which gene affects mostly.

Fig1.2: Mechanism of propionate generation

To control the bacterium’s generation of propionate. We use a bio-oscillator. (The design is shown in figure1.3) We firstly use an mRFP as reporter to test and verify our design.

Figure1.3: Design of our bio-oscillator

After finishing these two main works,we will replace mRFP with our key gene.Regulating the period of oscillator utilizing the frequency divider with an ssrA-tag analog attached to the end of enzyme.
We believe our oscillating BP reliever is both helpful and practical.

Results

As we divided our project to two main parts: propionate generation and oscillator. The results will be demonstrated from these two sections.

Propionate generation:

1.We firstly successfully copied the four genes ygfD/ygfH/ygfG/Sbm from E.coli strain K12, and then recombined them independently to expression vector. Purified protein from cell disruptions by Ni-chelating and did gel analysis. From figure-1 , we confirmed that the enzymes were successfully expressed.

Figure1. SDS-PAGE analysis of Sbm, ygfD, ygfG, ygfH over-expression

2.As we thought the overexpression of enzymes didn’t directly prove the increase of propionate, and we wanted to find the key gene in the reaction. So we used the positive clones to do fermentation. We firstly optimized the condition and found the best sampling time points. Then we used standard propionate of seven gradients to draw a linear graph between propionate concentration and HPLC peak area. Figure-2 shows the relationship.

Figure-2. Standard curve of propionate and HPLC peak area

Later, we used the recombinant strain to do fermentation, and measured propionate concentration in the samples. Figure-3 shows the propionate increase percent of each gene recombinant strain. From the data, we found propionate production had a significant increase when ygfD transformed. In other words, we found the key gene- our oscillator’s output.

Figure-3. HPLC analysis wild-type BL21 and recombination BL21 with four genes

Oscillator:

1.As for the oscillator, we first successfully constructed the two plasmids formed the dual-feedback circuit. (Circuit and plasmid were show in figure-4) LAA tag was added to the protein for rapid degradation. Gene sequencing confirmed the plasmids to be correct without any lethal mutation.

Figure-4.The dual-feedback circuit and two plasmids we constructed

2.After that, we transformed recombinant plasmid pET-28a (+) which had an mRFP reporter to check if it could function well. We induced the positive clone with 2mM IPTG and observed by fluorescence microscope. Figure-5 shows that it function well.

Figure-5. Fluorescence microscope photo of recombinant plasmid pET-28a (+) transformed cell induced by 2mM IPTG

3.Later, we co-transformed two recombinant plasmids pET-28a (+) and pACYCDuet-1. After inducing positive clone by 0.7% Arabinose and 2 mM IPTG, we used fluorescence microscope to see RFP change of single cell and used fluorospectro photometer to see RFP change of multicells.Figure-6 & Figure-7 shows the result.

Figure-6. Fluorescence change of single cell

Cells were diluted with culture medium and immobilized with glycerol. (5ul bacteria +20ulmedium+20ul glycerol). Making sure that the cell was alive and motionless, we could take photo of the same cell. From the pictures, we can see that the fluorescence changed with time in an oscillatory way, which supports our bio-oscillator design.

Figure-7. Fluorescence change of multicell

We used fluorospectro photometer to measure the oscillating behavior of multi-cells. After induced by 0.7% arabinose and 2 mM IPTG when OD600 was 0.55, cells were cultured in 37℃/200rpm.We sampled a series of time points and draw the curve as figure-7. We saw a significant fluorescence oscillating in compare to control group. The control group cells transformed pET-28a (+) only.

Summarization: We have successfully found a way to enhance cells generation of propionate, tested and verified our design of bio-oscillator. Combining these two works, we believe we can build a gut probiotic which can release propionate periodically in accord with the rhythm of human BP.

Future work

We do not have enough time to fulfill the whole project. Based on the pre-existing work, these tasks are coming to be finished in future.
1.Replace the report gene mRFP by ygfD in the dual-feedback circuit to see whether the propionate generation can oscillate.
2.Regulate the period of propionate generation to cope with human’s BP rhythm. We may utilize the frequency divider with an ssrA-tag analog attached to the end of enzyme to achieve it.

3.Transform the regulatory net into bifidobacterium – microorganism has reputation among the dairy industry — due to people who we sent questionnaire to show preference eating food containing probiotics rather than E.coli, Also, we will measure the propionate outside of the human body.

Judging Critieria

Already registered in the official website in 13th March and was accepted in 12th April.
1.We completed safety form, judging form and team wiki before the deadline. It is for sure that we are going to present a poster and a talk at the iGEM Jamboree.
2.We documented four newly standard BioBrick Part(sbm/ygfG/ygfH/ygfD) used in our project and submitted them to the iGEM Registry adhere to guidelines.
3.Our works aims at maintaining the blood pressure through microbe metabolism SCFA, which is a new application in medicine to our knowledge.
4.We did plenty of experiment to validate that two of BioBrick Part of our own design and construction works as expected.
5.We share information and material with WHU and HZAU .Cooperating with HZAU on characterizing one part.
6.We originaly creat a crossword to popularize historical knowledge about iGEM. That's a good new approach for human practice.
Therefore, we believe that we deserve a Gold Medal Prize.