Team:HUST-China/Project

From 2013.igem.org

(Difference between revisions)
Line 73: Line 73:
        </ul>
        </ul>
  </div>
  </div>
-
  <div class="span9" style="padding-left:20px">
+
    <div class="span9" style="padding-left:20px">
              <div id="scrollspy">
              <div id="scrollspy">
-
             <div id="abstract">
+
             <div id="Part-1">
-
             <h1 class="page-header">Abstract</h1>
+
             <h1 class="page-header"><strong>Part 1</strong>
-
             <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>
+
            <div style="font-size:28px" >The standardization of four genes</div></h1>
-
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>
+
             <p>
-
<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.  
+
            <img src="./images/project-figure1.png"width="500px;" style="float:right;"/>
 +
            We abstracted the target genes from the genome in E.coli K12 by PCR. However,there is one restriction enzyme site in ygfD that will has itself segmented without proper mutation.So we decided to obliterate the restriction site PstI by site-directed mutagenesis based on overlap extension PCR.After being sequenced,the four genes were linked to pmd18-T vector<span style="color:#232399">(Figure 1) </span>for amplification and preservation. <br>
 +
In addition, we utilized an expression vector 2118CA <span style="color:#232399">(Figure 2)</span> to make genes express in the DH5-α. By measuring the propionate production, we can know whether they truly work in the recombination vectors.<br>
 +
As iGEM officials require, the four genes are all standardized by adding EcoRI and PstI restriction enzyme sites at both terminates. They are all carried by psb1C3 vector<span style="color:#232399">(Figure 3)</span>.<br>
 +
             </p>
             </p>
 +
            <div class="row-fluid">
 +
            <div class="span6">
 +
              <p><img src="./images/project-figure2.png" width="343px"/></p>
 +
              <p class="small">Figure 2 : The 2118CA vector </p>
 +
            </div>
 +
            <div class="span6">
 +
              <p><img src="./images/project-figure3.png" width="343px"/></p>
 +
              <p class="small">Figure 3 : The psb1C3 vector</p>
 +
            </div>
 +
        </div>
             </div>
             </div>
-
             <div  id="overview">
+
             <div  id="Part-2">
-
             <h1 class="page-header">Overview</h1>
+
             <h1 class="page-header"><strong>Part 2</strong>
 +
            <div style="font-size:28px">The construction of the biological oscillator</div></h1>
            <p>
            <p>
-
            <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;"/>
+
            <div style="float:right;">
-
<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>
+
            <img src="./images/project-figure4.jpg" width="350px;" />
-
 
+
            <p class="small">Figure 4 : The structure of pET28-a</p>
 +
            </div>
 +
            According to Jesse Stricker, they described an engineered genetic oscillator in Escherichia coli that is fast, robust and persistent. The oscillator consists two parts, activator with the reporter gene rfp, and repressor. <br>
 +
In our project, the activator and reporter modules are on a derivative of 2118CA whose original promoter is replaced by hybrid promoter (plac/ara-1) , which will be activated by AraC in the presence of arabinose and repressed by LacI in the absence of IPTG. While the repressor module is on a derivative of pET28-a <span style="color:#232399">(Figure 5)</span>.with its own LacI gene destroyed and a new LacI gene inserted. In accordance with the activator, the original promoter is also replaced by plac/ara-1. <br>
 +
We supposed that if the fluorescence protein changes periodically, then we can substitute our target genes for rfp.<br>
 +
<em  style="font-size:11px;color:rgb(15, 7, 107)">
 +
<strong >Reference:</strong>
 +
Jesse Stricker, Scott Cookson, Matthew R. Bennett , William H. Mather, Lev S. Tsimring & Jeff Hasty. A fast, robust and tunable synthetic gene oscillator. Vol 456| 27 November 2008| doi:10.1038/nature07389
 +
</em>
            </p>
            </p>
            </div>
            </div>
-
             <div style="font-style:italic;font-size:11px;color:rgb(15, 7, 107)">
+
             <div id="Part-3">
-
            <strong>Reference</strong>
+
            <h1 class="page-header"><strong>Part 3</strong>
 +
            <div style="font-size:28px">The output evaluation of the propionate before and after the genes regulation</div></h1>
            <p>
            <p>
-
            <ol>
+
            To prove the efficiency of gene regulation, the propionate generated in recombination E.coli BL21 strain as well as the wild type E.coli BL21 strain are measured by HPLC analysis. According to the growth curve of wild type BL21 under the 37℃, we found the optimum time to sample is 18h after the fermentation sets out. Cell growth was monitored as the optical density at a wavelength of 600 nm (OD600). At first, we did some experiment to explore better substrate for fermentation besides Luria- Bertani (LB) broth. We added glucose, succinate, vitamin B12 to LB broth respectively, and then measure the centrifugal supernatant after 18h. We use Kromasil reverse-phase C18 column (250X4.6mm, 5μm) under the conditions that are listed below:
-
            <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>
+
            <dl class="dl-horizontal">
-
            <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>
+
  <dt>Flow rate:</dt><dd> 0.8ml/min</dd>
-
            <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>
+
  <dt>Mobile phase:</dt><dd> methanol : 0.005%KH2PO4(pH2.7)=0.1:0.9</dd>
-
            <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>
+
  <dt>Wavelength:</dt><dd> 215nm</dd>
-
            </ol>
+
</dl>
-
            </p>
+
</p>
            </div>
            </div>
-
              </div>
+
              </div>
-
  </div>
+
    </div>
-
  </div>
+
</div>
</div>
Line 132: Line 155:
     var scroll = $(document).scrollTop();
     var scroll = $(document).scrollTop();
     var navli = $("#navbar .nav li");
     var navli = $("#navbar .nav li");
-
var abstract = $("#abstract").outerHeight();
+
var part1= $("#Part-1").outerHeight();
-
var overview = $("#overview").outerHeight();
+
                                var part2= $("#Part-2").outerHeight();
-
+
                                var part3= $("#Part-3").outerHeight();
 +
var futurework= $("#Future-Work").outerHeight();
 +
                                var judge= $("#Judging-Criteria").outerHeight();
if(scroll > 454){
if(scroll > 454){
                                         $("#up").slideDown("ease-out");
                                         $("#up").slideDown("ease-out");
                                         $("#mainNavBar").addClass("navbar-fixed-top");
                                         $("#mainNavBar").addClass("navbar-fixed-top");
$(".bs-docs-sidenav").removeClass("affix-top").addClass("affix");
$(".bs-docs-sidenav").removeClass("affix-top").addClass("affix");
-
if (scroll < 454 + abstract ){
+
if (scroll < 454 + part1){
navli.removeClass("active").eq(0).addClass("active");
navli.removeClass("active").eq(0).addClass("active");
-
}else if (!(scroll < 454 + abstract || scroll > 454 + abstract + overview)){
+
}else if (!(scroll < 454 + part1|| scroll > 454 + part1+ part2)){
navli.removeClass("active").eq(1).addClass("active");
navli.removeClass("active").eq(1).addClass("active");
 +
}else if (!(scroll < 454 + part1 + part2 || scroll > 454 + part1+ part2 + part3)){
 +
navli.removeClass("active").eq(2).addClass("active");
 +
}else if (!(scroll < 454 + part1 + part2 + part3 || scroll > 454 + part1+ part2 + part3 + futurework)){
 +
navli.removeClass("active").eq(3).addClass("active");
 +
}else if (!(scroll < 454 + part1 + part2 + part3  + futurework || scroll > 454 + part1+ part2 + part3 + judge)){
 +
navli.removeClass("active").eq(4).addClass("active");
}
}
}else{
}else{

Revision as of 20:08, 21 September 2013

Part 1
The standardization of four genes

We abstracted the target genes from the genome in E.coli K12 by PCR. However,there is one restriction enzyme site in ygfD that will has itself segmented without proper mutation.So we decided to obliterate the restriction site PstI by site-directed mutagenesis based on overlap extension PCR.After being sequenced,the four genes were linked to pmd18-T vector(Figure 1) for amplification and preservation.
In addition, we utilized an expression vector 2118CA (Figure 2) to make genes express in the DH5-α. By measuring the propionate production, we can know whether they truly work in the recombination vectors.
As iGEM officials require, the four genes are all standardized by adding EcoRI and PstI restriction enzyme sites at both terminates. They are all carried by psb1C3 vector(Figure 3).

Figure 2 : The 2118CA vector

Figure 3 : The psb1C3 vector

Part 2
The construction of the biological oscillator

Figure 4 : The structure of pET28-a

According to Jesse Stricker, they described an engineered genetic oscillator in Escherichia coli that is fast, robust and persistent. The oscillator consists two parts, activator with the reporter gene rfp, and repressor.
In our project, the activator and reporter modules are on a derivative of 2118CA whose original promoter is replaced by hybrid promoter (plac/ara-1) , which will be activated by AraC in the presence of arabinose and repressed by LacI in the absence of IPTG. While the repressor module is on a derivative of pET28-a (Figure 5).with its own LacI gene destroyed and a new LacI gene inserted. In accordance with the activator, the original promoter is also replaced by plac/ara-1.
We supposed that if the fluorescence protein changes periodically, then we can substitute our target genes for rfp.
Reference: Jesse Stricker, Scott Cookson, Matthew R. Bennett , William H. Mather, Lev S. Tsimring & Jeff Hasty. A fast, robust and tunable synthetic gene oscillator. Vol 456| 27 November 2008| doi:10.1038/nature07389

Part 3
The output evaluation of the propionate before and after the genes regulation

To prove the efficiency of gene regulation, the propionate generated in recombination E.coli BL21 strain as well as the wild type E.coli BL21 strain are measured by HPLC analysis. According to the growth curve of wild type BL21 under the 37℃, we found the optimum time to sample is 18h after the fermentation sets out. Cell growth was monitored as the optical density at a wavelength of 600 nm (OD600). At first, we did some experiment to explore better substrate for fermentation besides Luria- Bertani (LB) broth. We added glucose, succinate, vitamin B12 to LB broth respectively, and then measure the centrifugal supernatant after 18h. We use Kromasil reverse-phase C18 column (250X4.6mm, 5μm) under the conditions that are listed below:

Flow rate:
0.8ml/min
Mobile phase:
methanol : 0.005%KH2PO4(pH2.7)=0.1:0.9
Wavelength:
215nm

Acknowledgement

CopyRight ©2013.igem.org All Rights Reserved!
Powered by the iGEM team comes from HuaZhong University of Science and Techology  HUST-China

Retrieved from "http://2013.igem.org/Team:HUST-China/Project"