Team:HUST-China/Project

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                 <div id="Overview">
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             <h1 class="page-header"><strong>Overview</strong></h1>                                       
             <h1 class="page-header"><strong>Overview</strong></h1>                                       
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            <p> <div style="font-size:28px;line-height:30px;margin:10px 0;">Hypertension</div> is an important worldwide public-health challenge .It has been identified as the leading risk factor for mortality mainly because it can lead to adverse cardiovascular events . It has already affects one billion people through out world and kills nine million people each year. And the incidence of it appears to follow a circadian pattern, reaching a peak in the morning shortly after wakening and arising. <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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It’s not possible to take pills when asleep.
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<div style="text-align:center">
<a href="https://static.igem.org/mediawiki/2013/e/ea/HUST-projectoverview1.jpg" target="_blank">           
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<p><em style="font-size:11px;">Figure1.1: human blood pressure daily rhythm</em></p>
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There is a wide selection of antihypertensive drugs designed for patients. However, traditional treatment comes along with heavy financial burden and severe side effects along with drug dependence. To make the matter worse, these medicines can not stop morning surge taking lives from patients.<br>
 
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Short chain fatty acid(SCFA) ,especially acetate and propionate, was newly proved to cause  an acute hypotensive response. A GPCRs called olfr78 expressed in smooth muscle cells of small blood vessel plays an important role . It could be activated by SCFA and induce vasodilatation and hypotension. In consideration of the case, we developed a novel method to treat hypertension.By utilizing a group of friendly engineering bacteria which can release propionate piriodcally in the human intestine. <br>
 
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To achieve this,we found a four enzyme pathway in E.coil that converts succinate to propionate. By combining key genes and bio-oscillator together, we try to make E.coli release propionate 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. In that case, patients will not worry about excessive surge anymore.<br>
 
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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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By conducting this project, we hope to provide a brand new way for those patients suffering from hypertension to live a healthy life.
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<img src="https://static.igem.org/mediawiki/2013/c/c0/HUST-projectoverview4.png"/>
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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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             <div  id="Results">
             <div  id="Results">
             <h1 class="page-header"><strong>Results</strong></h1>
             <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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                                                1 .Biobricks (we submit / Registry ) table describe<br>
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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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            <caption>Table 3-11: Information of four standard biobricks</caption>
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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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      <th>Standard biobricks </th>
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      <td>pSB1C3-ygfD(EocRI PstI)</td>
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      <td>According to Toomas Haller, the enzyme 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.</td>      
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      <td>pSB1C3-ygfH(EocRI PstI)</td>
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      <td>This gene encode propionyl-CoA:succinate CoA transferase that catalyzes a CoA transferase reaction from propionyl-CoA to succinyl, generating propionate.</td>
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      <td>pSB1C3-ygfG(EocRI PstI)</td>
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      <td>The third gene in the operon encoding methylmalonyl-CoA decarboxylase that catalyzes the decarboxylation of methylmalonyl-CoA to propionyl-CoA</td>
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      <td>pSB1C3-sbm(EocRI PstI) </td>
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      <td>Sbm encodes methylmalonyl-CoA epimerase which catalyzes the reversible reaction of succinyl-CoA and methylmalonyl -CoA</td>
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2 .We successfully constructed the four genes on expression vector. Than transformed them independently into E.coli BL21. After found most suitable medium and built our own measurement method by HPLC.we found that propionate’s throughput can rise over 10% with ygfD and sbm transformed, yet over 5% rising for ygfH and ygfG. We successfully found a way to increase the output of propionate. And confirm that ygfD is the best for the oscillator.<br>
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<img src="https://static.igem.org/mediawiki/2013/9/95/HUST_HPLC.png" /><br>
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3.As to the oscillator device, the dual-feedback circuit driven by the hybrid promoter was successfully constructed.
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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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<img src="https://static.igem.org/mediawiki/2013/4/48/HUST_PACYCDuet-1.png" style="width:343px;height:270px"/></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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But we still did not observe the periodically changing fluorescence during the fermentation.To testify the hypothesis of the modeling team, we add LAA-tag to each gene in the oscillator device, to extended the period to 1 hour. Later we will use confocal and FCM to detect the fluorescence. The results will be show in our presentation. And we hope time is enough for us to do live cell imaging. What we expect could be like the video.<a href="https://static.igem.org/mediawiki/2013/7/7b/HUST_Oscillating_Cells.mov">Click here to view.</a><br>
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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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</div>
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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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</div>
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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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             <div  id="Future-Work">
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             <h1 class="page-header"><strong>Future work</strong></h1>
             <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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            There is a big challenge in our project that the recombination microbe has to be validated in the human intestine. Based on the pre-existing work, a plenty of works are coming to us in the near 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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1.Regulating the period of propionate utilizing the frequency divider with a ssrA-tag analog attached to the end of enzyme.<br>
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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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2. Replace the report gene rfp encoding red fluorescence protein with key gene encoding enzymes in the synthetic pathway. <br>
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3. As we know that E.coli is the most popular chassi used in the synthetic biology. But according to the people we sent questionnaire to, they prefer eating food containing probiotics rather than eating bacteria. So we are going to transform the regulatory net into bifidobacterium, which enjoy a highly reputation among the dairy industry. We will measure the propionate outside of the human body.<br>
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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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4. Using mathematical modeling to imitate the environment in the intestine and the concentration decreasing of propionate in the blood circulation. <br>
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<strong>Acknowledgement</strong>
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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.