Team:Tokyo Tech/Projects/Urea-cooler/index.htm

From 2011.igem.org

(Difference between revisions)
 
(169 intermediate revisions not shown)
Line 18: Line 18:
top: 500px;
top: 500px;
position: static;
position: static;
 +
font-size: 12px;
}
}
Line 34: Line 35:
list-style: none;
list-style: none;
float: left;
float: left;
-
height: 20px;
+
height: 30px;
-
width: 732px;
+
width: 945px;
}
}
Line 43: Line 44:
position:relative;
position:relative;
padding: 2px 15px 0 0;
padding: 2px 15px 0 0;
-
/* border-color: #EFEBEC;
 
-
border-width: 0;
 
-
border-left-width: 5px;
 
-
border-style: solid; */
 
}
}
Line 55: Line 52:
display: none;
display: none;
position: absolute;
position: absolute;
-
font-size: 14px;
+
font-size: 16px;
opacity: 0.8;
opacity: 0.8;
list-style: none;
list-style: none;
Line 79: Line 76:
background-color: #EFEBEC;
background-color: #EFEBEC;
color: #000000;
color: #000000;
 +
font-size: 13px;
}
}
Line 86: Line 84:
left: 0;
left: 0;
clear: both;
clear: both;
-
height: 180px;
+
height: 190px;
}
}
.main
.main
Line 106: Line 104:
h1, h2, h3, h4, h5, h6
h1, h2, h3, h4, h5, h6
{
{
-
background-color: #D09DE1;
+
background-color: #00508D;
-
color:#0D3430;
+
color:#FFFFFF;
text-align: left;
text-align: left;
}
}
Line 114: Line 112:
{
{
text-indent: 1em;
text-indent: 1em;
-
}
 
-
 
-
h2+p
 
-
{
 
-
margin-left: 1em;
 
}
}
Line 124: Line 117:
{
{
text-indent: 2em;
text-indent: 2em;
-
}
 
-
 
-
h3+p
 
-
{
 
-
margin-left: 2em;
 
}
}
Line 134: Line 122:
{
{
text-indent: 3em;
text-indent: 3em;
 +
}
 +
 +
h5
 +
{
 +
text-indent: 4em;
}
}
-
h4+p
+
h6
{
{
-
margin-left: 3em;
+
text-indent: 5em;
}
}
-
+
.footer
.footer
{
{
Line 164: Line 157:
}
}
-
p
+
.gene
{
{
-
text-indent: 0.5em;
+
font-style: italic;
}
}
 +
p
 +
{
 +
text-indent: 2em;
 +
}
 +
.graph_title
.graph_title
{
{
Line 175: Line 173:
}
}
 +
table
 +
{
 +
min-width: 150px;
 +
align: center;
 +
}
-->
-->
</style>
</style>
Line 215: Line 218:
}
}
-
window.onload = function() {
+
function getElementsByClass() {
 +
    var classElements = new Array();
 +
    var allElements = document.getElementsByTagName("*");
 +
    for (i = 0 ; i < allElements.length; i++) {
 +
if (allElements[i].className == 'firstHeading') {
 +
    allElements[i].style.display = 'none';
 +
  // window.alert('発見。');
 +
}
 +
    }
 +
}
 +
 +
window.onload = function() {
 +
getElementsByClass();
var menu = window.document.getElementById('TopMenu');
var menu = window.document.getElementById('TopMenu');
if(menu==null)
if(menu==null)
Line 248: Line 263:
<!-- top box -->
<!-- top box -->
<div class="top">
<div class="top">
-
<img src="https://static.igem.org/mediawiki/2011/2/24/TokyoTech_TOPlogo.png" alt="Tokyo Tech 2011" /><br />
+
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=6,0,0,0" width="965" height="150" id="Yourfilename" align="">
 +
<param name="movie" value="https://static.igem.org/mediawiki/2011/4/4d/Header.swf">
 +
<param name="quality" value="high">
 +
<param name="bgcolor" value="#FFFFFF">
 +
<embed src="https://static.igem.org/mediawiki/2011/4/4d/Header.swf" quality="high" bgcolor="#FFFFFF" width="965" height="150" name="Yourfilename" align="" type="application/x-shockwave-flash" pluginspage="http://www.macromedia.com/go/getflashplayer">
 +
</embed>
 +
</object>  
<!-- list of top menu -->
<!-- list of top menu -->
<div id="navigation">
<div id="navigation">
Line 255: Line 276:
<li id="menu_Project">
<li id="menu_Project">
-
Projects
+
Project
<ul>
<ul>
-
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/index.htm">RPS-game</a></li>
+
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/index.htm">RPS-Game</a></li>
-
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/making-rain/index.htm">rain</a></li>
+
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/making-rain/index.htm">Make it Rain</a></li>
-
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/Urea-cooler/index.htm">urea cooler</a></li>
+
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/Urea-cooler/index.htm">Urea Coolers</a></li>
</ul>
</ul>
</li>
</li>
Line 268: Line 289:
Modeling
Modeling
<ul>
<ul>
-
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/RPS-game/PRS-game">RPS-game</a></li>
+
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/RPS-game/RPS-game">RPS-Game</a></li>
-
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/urea-cooler">urea cooler</a></li>
+
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/urea-cooler">Urea Coolers</a></li>
</ul>
</ul>
</li>
</li>
Line 278: Line 299:
<li id="menu_Extra">
<li id="menu_Extra">
-
Extra
+
More
-
<ul>
+
<ul style="width:210px;">
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Safety">Safety</a></li>
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Safety">Safety</a></li>
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Attribution_and_Contributions.htm">Attribution and Contributions</a></li>
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Attribution_and_Contributions.htm">Attribution and Contributions</a></li>
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/notebook">NoteBook</a></li>
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/notebook">NoteBook</a></li>
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/team">Team</a></li>
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/team">Team</a></li>
-
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Sponsers.htm">Sponsers</a></li>
+
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Sponsers.htm">Sponsors</a></li>
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Collaboration.htm">Collaboration</a></li>
<li><a href="https://2011.igem.org/Team:Tokyo_Tech/Collaboration.htm">Collaboration</a></li>
</ul>
</ul>
Line 298: Line 319:
<!-- left menu list -->
<!-- left menu list -->
-
<div style="min-height: 3500px; float: left;">
+
<div style="min-height: 10700px; float: left;">
<div id="LeftMenu">
<div id="LeftMenu">
<!--list of page menu: DO NOT WRITE LINKS NOT WRITTEN IN THIS PAGE -->
<!--list of page menu: DO NOT WRITE LINKS NOT WRITTEN IN THIS PAGE -->
<ul>
<ul>
<li>
<li>
-
<a href="#1.abst">1. Abstruct</a>
+
<a href="#abst">1. Abstruct</a>
 +
                </li>
 +
                <li>
 +
                        <a href="#2.Gene">2. Genetic Engineering for Urea Production</a>
<ul>
<ul>
<li><a href="#2.1Intro">2.1 Introduction</a></li>
<li><a href="#2.1Intro">2.1 Introduction</a></li>
-
<li><a href="#2.2">2.2 Result</a></li>
+
<li><a href="#2.2">2.2 Charcterization of <i>roc</i>F and Arg box</a></li>
 +
                                <li><a href="#2.3">2.3 Characterization of Ptrc-RBS-<i>rocF</i>-Arg box</a></li>
 +
                                <li><a href="#2.4">2.4 Use of mutation in <i>argR</i> gene</a></li>
</ul>
</ul>
 +
               
</li>
</li>
 +
                <li>
 +
                        <a href="#Flux">3. Flux analysis for providing more urea </a>
 +
                        <ul>
 +
                                <li><a href="#abstract">3.1 Abstract</a></li>
 +
                                <li>
 +
                                            <a href="#intro">3.2 Introduction</a>
 +
                                            <ul>
 +
                                                        <li><a href="#modes">3.2.1 What is Elementary Flux Modes?</a></li>
 +
                                                        <li><a href="#analyzing">3.2.2 Analyzing the function of the compounds involved in the Urea Cycle by determining the elementary flux modes</a></li>
 +
                                                        <li><a href="#finding">3.2.3 Finding Modes to Increase the Urea production by <i>E. coli</i></a></li>
 +
                                            </ul>
 +
                                </li>                           
 +
                                <li>
 +
                                            <a href="#results">3.3 Results</a>
 +
                                            <ul>
 +
                                                          <li><a href="#funciton">3.3.1 Analyzing the function of the compounds involved in the Urea Cycle by determining the elementary flux modes</a></li>
 +
                                                          <li><a href="#production">3.3.2 Finding Modes to Increase the Urea production by <i>E. coli</i></a></li>
 +
                                            </ul>
 +
                                </li>
 +
                                <li><a href="#future">3.4 Future Work</a></li>
 +
                        </ul>
 +
                </li>
</ul>
</ul>
</div>
</div>
Line 315: Line 364:
<!-- main contents -->
<!-- main contents -->
<div class="main">
<div class="main">
-
<!-- ############ Wrote main contents here ############### -->
+
<!-- ############ Write main contents here ############### -->
<!-- page title -->
<!-- page title -->
<h1> Urea cooler </h1>
<h1> Urea cooler </h1>
-
<p>
+
-
<h2 id="1.abst">1. Abstract</h2>
+
<h2 id="abst">1. Abstract</h2>
<p>
<p>
-
We made urea cycle in E.coli by introducing of arginase encoded by rocF gene  
+
Coolers can be made by dissolving urea in water because it is an endothermic reaction. So, in order to obtain urea, we made the urea cycle in<i> E.coli</i> by introducing <i>rocF</i> gene encoding arginase, which is an enzyme that converts L-arginine to L-ornithine and urea. But just by introducing <i>rocF</i>, only a little amount of urea can be produced because arginine biosynthesis is repressed by the arginine repressor. Therefore, we tried to derepress arginine biosynthesis in two ways. One is by introducing arginine operator sequences(Arg boxes), which bind the arginine repressor. The other is by using a strain that carries a mutation in a gene which encodes arginine repressor. Furthermore, we studied elementary flux modes to provide more urea. As a result, we found out that the artificial urea production system is robust in a stoichiometric point of view. The analysis also revealed that supplementation of arginine, glutamic acid and aspartic acid would increase urea production rate.<br />
-
and get urea to make urea cooler. To make urea cooler,
+
<table style="text-align:center;" align="center">
-
we need large amount of urea. But just by introducing rocF,  
+
<tr>
-
only a little amount of urea can be produced because arginine biosynthesis is  
+
<td>
-
repressed. Therefore, we tried to derepress the effect of repression.<br />
+
<img src="https://static.igem.org/mediawiki/2011/c/cb/Arg_box_on_1C3.png" alt="Assay data" width="350px" align="center" />
-
Furthermore, we researched flux to provide more urea.  
+
</td>
-
As a result, we found that the artificial urea production system,
+
<td>
-
as well as natural one, is robust in a stoichometrically
+
<img src="https://static.igem.org/mediawiki/2011/e/e7/TokyoTech_Urea-fig5.png" alt="Fig5" width="350px" />
-
point of view. The analysis also found that supplementation of Arg,  
+
</td>
-
Glu and Asp would increase urea production rate.<br />
+
</tr>
-
<img src="https://static.igem.org/mediawiki/2011/c/cb/TokyoTech_urea-assay1.png" alt="Assay data" />
+
<tr>
-
<div class="graph_title">
+
<td>
-
Fig.  The reactions related with the urea cycle
+
Fig. 3 Urea concentration in growth media 1 hour after IPTG induction  
-
Fig.4 Urea concentration in growth media 1 hour after IPTG induction
+
</td>
-
</div>
+
                                <td>
 +
Fig. 5  The reactions related with the urea cycle
 +
</td>
 +
</tr>
 +
</table>
</p>
</p>
 +
                <h2 id="2.Gene">2. Genetic Engineering for Urea Production</h2>
<h3 id="2.1Intro">2.1 Introduction</h3>
<h3 id="2.1Intro">2.1 Introduction</h3>
<p>
<p>
-
Coolers can be made by adding urea to water, since dissolving urea in water  
+
Coolers can be made by dissolving urea in water, since dissolution of urea in water is an endothermic reaction (-57.8 cal/g). So we came up with an idea of creating <i>E. coli</i> that can synthesize urea. Originally, <i>E. coli</i> has all the enzymes in the urea cycle except for arginase, which converts L-arginine into L-ornithine and urea(Fig. 1). So we attempted to complete the urea cycle in <i>E. coli</i> by introducing a gene which encodes arginase and obtain urea.<br />
-
is an endothermic reaction (-57.8 cal/g). However, E. coli does not synthetize
+
In this work, introduction of the <i>Bacillus subtilis</i> <i>rocF</i> gene which encodes arginase on a standardized plasmid completed urea cycle and enabled<i> E.coli</i> to produce urea as reported by TUCHMAN <i>et al</i>.,(1997).
-
urea naturally, so we attempted to complete the urea cycle inside E. coli and  
+
</p>
-
get urea. <br />
+
-
Originally, E.coli has all enzymes of the urea cycle except for the arginase.
+
-
In this work, introduction of the Bacillus subtilis rocF gene on a  
+
-
standardized plasmid completed urea cycle and enabled E.coli to produce urea  
+
-
as reported by TUCHMAN et al., (1997)  <br />
+
-
(Fig.1).  
+
-
<img src="https://static.igem.org/mediawiki/2011/2/21/TokyoTech_urea-cycle1.png" alt="Urea cycle; Fig1" />
+
-
+
-
<div class="graph_title">
+
-
Fig.1 Addition of a gene which codes arginase completes urea cycle in E.coli
+
-
</div>
+
-
However, just by introducing arginase , E.coli, has showed to produce only
+
<center>
-
a little amount of urea. TUCHMAN et al proposed that catabolite repression in
+
<img src="https://static.igem.org/mediawiki/2011/2/21/TokyoTech_urea-cycle1.png" alt="Urea cycle; Fig1"  width="700px" align="center" />
-
arginine biosynthesis pathway is the main reason for the low production
+
<div class="graph_title">
-
efficiency(TUCHMAN et al., 1997) The bacterial arginine biosynthetic genes  
+
Fig. 1 Addition of a gene which encoding arginase completes urea cycle in <i>E.coli</i>.
-
are all regulated via a common repressor protein encoded by the argR gene and activated  
+
</div>
-
in the presence of arginine . (Fig.3)They circumvented the arginine
+
</center>
-
repression by introduction of arginine operator sequences (Arg boxes),  
+
-
which bind the arginine repressor.  Upon arginine repressor binding to  
+
<p>
-
Arg boxes, the amount of the arginine repressor which can repress arginine  
+
However, just by introducing arginase, <i>E. coli</i> produces
-
biosynthesis is reduced.  In this work, we tried two ways of solving this
+
only a little amount of urea. TUCHMAN <i>et al</i>. proposed that catabolite  
-
problem. One way is introducing the Arg boxes as previous work.  
+
repression in arginine biosynthesis pathway is the main reason for  
-
The other way is using an E. coli that has an argR deletion genotype so  
+
the low efficiency of production(TUCHMAN <i>et al</i>., 1997). The bacterial  
-
that the repressor is not synthetized.   
+
arginine biosynthetic genes are all regulated via a common repressor  
 +
protein (arginine repressor) encoded by the <i>argR</i> gene. The arginine represor is activated in the presence of arginine (Fig. 2).<br/>
 +
TUCHMAN <i>et al</i>. circumvented the repression by introduction of  
 +
arginine operator sequences (Arg boxes), which bind the arginine repressor.   
 +
With the arginine repressor binding to Arg boxes, the amount of the arginine  
 +
repressor which can repress arginine biosynthesis is reduced.   
 +
In this work, we tried two ways of derepressing arginine biosynthesis. One way  
 +
is by introducing the Arg boxes as previous work. The other way is by using an  
 +
<i>E. coli</i> strain that has an <i>argR</i> deletion genotype so that
 +
the repressor is not synthetized.   
 +
</p>
-
<img src="https://static.igem.org/mediawiki/2011/9/98/TokyoTech_urea-arg-biosynth.png" alt="Fig2" />
+
<center>
-
<div class="graph_title">
+
<img src="https://static.igem.org/mediawiki/2011/3/30/Arginine_repressor.png" alt="Arginine biosynthesis is repressed by arginine repressor in the presence of arginine." align="center" width="600px"/>
-
Fig.2 Arginine biosynthesis is repressed by arginine repressor and its co-repressor
+
<div class="graph_title">
-
</div>
+
Fig. 2 Arginine biosynthesis is repressed by the arginine repressor in the presence of arginine.
 +
</div>
 +
</center>
 +
 
 +
<h3 id="2.2">2.2 Charcterization of <i>rocF</i> and Arg box</h3>
 +
 
 +
                <p>
 +
                Introduction of <i>rocF</i> gene on our part <a href="http://partsregistry.org/Part:BBa_K649301">BBa_K649301</a> led to production of more urea compared to negative controls. Therefore, we confirmed that insertion of <i>rocF</i> gene resulted in arginase production and completed the urea cycle in <span class="name">E. coli</span> as expected.
 +
                </p>
 +
               
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2011/c/cb/Arg_box_on_1C3.png" align="center" width="400px"/>
 +
</center>
 +
                <div class="graph_title">
 +
Fig. 3 Urea concentration in growth media when <i>rocF</i> or Arg box were introduced strain MG1655.
 +
</div>
 +
</center>
 +
 
 +
                <p>
 +
As a way to derepress arginine biosynthesis we introduced Arg box on our part <a href="http://partsregistry.org/Part:BBa_K649401">BBa_K649401</a> in addition to <i>rocF</i> gene (<a href="http://partsregistry.org/Part:BBa_K649301">BBa_K649301</a>). This led to production of even more urea. These results show that Arg boxes are effectively derepressing arginine production by deactivating the arginine repressor. <a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/Urea-cooler/data#1.1">Detailed information is shown here</a>.
 +
</p>
 +
       
 +
               
 +
<h3 id="2.3">2.3 Characterization of Ptrc-RBS-<i>rocF</i>-Arg box</h3>
 +
<p>
 +
In this study Arg boxes and <i>rocF</i> were introduced separately on high-copy-number plasmids (pSB1C3) and low-copy-number plasmids (pSB3K3). We also tested the effect of Arg boxes when they were introduced downstream of <i>rocF</i> gene on low-copy-number plasmids (pSB3K3 and pSB6A1). Here, Arg boxes didn’t work effectively. This is probably because a low-copy-number plasmid is not capable of introducing enough number of Arg boxes to effectively deactivate the arginine repressor.<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/Urea-cooler/data#2.1"> Detailed information is shown here</a>.
</p>
</p>
-
<h3 id="2.2">2.2 Results</h3>
+
<h3 id="2.4">2.4 Use of mutation in <span class="gene">argR</span> gene</h3>
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2011/0/0b/RocF_on_3K3_%282%29.png" align="center" width="400px"/>
 +
                <div class="graph_title">
 +
Fig. 4 Urea concentration in growth media when <i>rocF</i> gene on pSB3K3 was introduced in strain MG1655 (<span class="gene">argR</span> +) or JE6852 (<span class="gene">argR</span> -).
 +
</div>
 +
</center>
 +
<p>
<p>
-
Bacterial strains and plasmids
+
As another way to derepress arginine biosynthesis, we used an <span class="name">E. coli</span> strain that has an <span class="gene">argR</span> deletion genotype (JE6852, This strain was obtained from National Institute of Genetics). In our assay results, much more urea was produced when <i>rocF</i> gene was introduced in JE6852 than in MG1655 as expected.<a href="https://2011.igem.org/Team:Tokyo_Tech/Projects/Urea-cooler/data#2.1"> See more details about this experiment.</a>
-
The bacterial strains and plasmids used in this study are listed in Table 1 and Table 2, and the constructions are shown in Fig.3.
+
                </p>
 +
 
 +
                <h2 id="Flux"> 3. Flux analysis for providing more urea </h2>
 +
<h3 id="abstract">3.1 Abstract </h3>
 +
<p>
 +
This section is about a metabolic engineering study we did about the urea cycle. On the first part we show how we used &ldquo;elementary flux modes&rdquo; (Schuster <i>et al</i>., 2000) to analyze the function of the compounds involved in the urea cycle. Mainly we deduced which compounds act as sources of carbon and sources of nitrogen for the production of urea. On the second part of this study we show how we determined elementary flux modes of the urea cycle to find ways to increase the yield of urea. We focused on a strategy which involves increasing the concentration of four components of the cycle and which we concluded would yield more urea. To confirm our results future experiments will be done.
 +
</p>
-
<table>
+
<div class="graph_title">
-
<caption>TABLE 1. E.coli strains used in this study</caption>
+
<img src="https://static.igem.org/mediawiki/2011/e/e7/TokyoTech_Urea-fig5.png" alt="Fig5" width="750px" /><br />
-
<tr>
+
Fig. 5 The reactions related with the urea cycle
-
<th>Strain</th>
+
</div><br />
-
<th>argR</th>
+
*The orange letters are the abbreviated names of the enzymes involved. The red letters are the enzyme expressed by introducing <span class="gene"><i>rocF</i></span> gene. For complete names of the enzymes see <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table3">Table 3</a>.
-
</tr>
+
 
-
<tr>
+
 
-
<td>MG1655</td>
+
<h3 id="intro">3.2 Introduction</h3>
-
<td>+</td>
+
<h4 id="modes">3.2.1 What is Elementary Flux Modes?</h4>
-
</tr>
+
<p>
-
<tr>
+
In Metabolic Engineering, mathematical modeling is an effective way to increase the products of a reaction. In particular, Flux Analysis, which is based on the hypothesis that the system is in a steady state, is effective to find how to increase these products. The concept of elementary flux mode provides a mathematical tool to define and comprehensively describe all metabolic routes that are both stoichiometrically and thermodynamically feasible for a group of enzymes. As a method of metabolic flux analysis, it is based on the hypothesis that the concentration of the reactants and products involved in the cycle does not change.
-
<td>JD24293</td>
+
</p>
-
<td>-</td>
+
 
-
</tr>
+
<h4 id="analyzing">3.2.2 Analyzing the function of the compounds involved in the Urea Cycle by determining the elementary flux modes</h4>
-
</table>
+
<p>
-
+
By determining the elementary flux modes of a cycle we can have a more clear view of the function of each of the compounds involved in the cycle being analyzed. Based on the elementary flux modes of the urea cycle, in this study we could deduce that HCO<sub>3</sub><sup>-</sup> acts as the source of carbon for urea production and that both L-glutamine and NH<sub>3</sub> act as nitrogen sources for the formation of urea.
-
JD24293 was obtained from National Institute of Genetics.
+
</p>
-
+
 
-
<table>
+
<center>
-
<caption>TABLE2. Expression plasmids used in this study</caption>
+
<img src="https://static.igem.org/mediawiki/2011/1/14/T%280%29.png" alt=T(0) width="200px" />
-
<tr>
+
<span style="font-size:12px;">&rarr;</span>
-
<th>Designation</th>
+
<img src="https://static.igem.org/mediawiki/2011/2/28/T9.png" alt=T9 width="200px" />
-
<th>vector</th>
+
<img src="https://static.igem.org/mediawiki/2011/8/83/Urea_modeling.png" alt="Fig7" width="400px" />
-
<th>rocF</th>
+
</center>
-
<th>Arg Box</th>
+
 
-
</tr>
+
<h4 id="finding">3.2.3 Finding Modes to Increase the Urea production by <i>E. coli</i></h4>
-
<tr>
+
<p>
-
<td>Ptrc-rocF</td>
+
In this study we determined elementary flux modes to maximize urea production by <i>E. coli</i>. We found that there are two main strategies to increase urea production: one is to increase the amount of carbamoyl phosphate (which formation is known to be the rate-limiting step of the urea cycle). The other one is to increase the concentration of four components of the urea cycle: L-ornithine, L-citrulline, N-(L-arginino)succinate and L-arginine. We deduced the latter strategy by determining the elementary modes of the urea cycle, and therefore in this study we will focus on the description of this strategy.
-
<td>pSB3K3</td>
+
</p>
-
<td>+</td>
+
 
-
<td>-</td>
+
<div class="graph_title">
-
</tr>
+
<img src="https://static.igem.org/mediawiki/2011/d/d6/Cars.png" alt=T(0) width="500px" /><br />
-
<tr>
+
Fig. 8 Two ways to increase urea production
-
<td>Ptrc-rocF-Arg Box</td>
+
</div>
-
<td>pSB3K3</td>
+
 
-
<td>+</td>
+
<h3 id=results>3.3 Results</h3>
-
<td>+</td>
+
 
-
</tr>
+
<p>
-
</table>
+
In our study, we considered the enzymatic reactions shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table3">Table 3</a> to determine the elementary flux modes related to urea production by <i>E. coli</i>. The scheme the overall reaction system is shown in Fig. 5 below.
-
   
+
</p>
-
<img src="https://static.igem.org/mediawiki/2011/6/67/TokyoTech_urea-parts-design.png" alt="plasmid map" />
+
-
<div class="graph_title">
+
<div class="graph_title">
-
Fig.3 Plasmids used in this study
+
<img src="https://static.igem.org/mediawiki/2011/e/e7/TokyoTech_Urea-fig5.png" alt="Fig5" width="600px" /><br />
-
The details of the constructions are here.
+
Fig. 5 The reactions related with the urea cycle
 +
</div>
 +
<p>
 +
*The orange letters are the abbreviated names of the enzymes involved. The red letters are the enzyme expressed by introducing <span class="gene">rocF</span> gene. For complete names of the enzymes see <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table3">Table 3</a>.
 +
</p>
 +
 
 +
<p>
 +
By determining the elementary flux modes to produce urea inside <i>E. coli</i>, we found two important results:
 +
</p>
 +
<ol>
 +
<li><p>
 +
We confirmed both L-glutamine and NH<sub>3</sub> act as nitrogen providers in the urea cycle, as well as deducing that HCO<sub>3</sub><sup>-</sup> acts as the source of carbon for urea production. These modes did not make use of organic intermediates. Even though L-glutamine is consumed in order to to transfer the side-chain ammonium group needed for the production of carbamoyl phosphate (which in turn transfers the ammonium group to the urea cycle), free ammonium ion can restore L-glutamine from L-glutamate (which is a byproduct of the reaction that yields carbamoyl phosphate as a product).
 +
</p></li>
 +
<li><p>
 +
We concluded that increasing the concentration of L-ornithine will increase the concentration of three related compounds (L-citrulline, N-(L-arginino)succinate, and L-arginine) and this will ultimately lead to an increase in the production of urea. We also noted that since the L-aspartate amino acid, which is needed in the urea cycle we considered(Fig. 5), is normally consumed in protein biosynthesis, so it should be supplied in the culture medium or synthetized by <span class="name">E. coli</span> in order to be able to increase the amount of urea and to maintain the cycles that produce it.
 +
</p></li>
 +
</ol>
 +
 
 +
<p>
 +
Below is a detailed description of these three results.
 +
</p>
 +
 +
<h4 id="funciton">3.3.1 Analyzing the function of the compounds involved in the Urea Cycle by determining the elementary flux modes</h4>
 +
<p>
 +
The first step was to determine the flux modes which need of L-glutamine as an input (Mendel <i>et al</i>., 1996). We did this by calculations based on a matrix as the tableau shown below.
 +
</p>
 +
 
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2011/1/14/T%280%29.png" alt=T(0) width="800px" /><br />
 +
<img src="https://static.igem.org/mediawiki/2011/2/28/T9.png" alt=T9 width="800px" />
 +
</center><br />
 +
 
 +
<a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/method" align="right">Details about the calculations can be found here</a>
 +
 
 +
<p>
 +
We found eight modes that can produce urea without using organic intermediates. These are shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/figures#Elem1">Fig. 6</a>. Each reaction formula is shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table4">Table 4</a>. In particular, we focused on one the mode displayed in Fig. 7.
 +
</p>
 +
 
 +
<div class="graph_title">
 +
<img src="https://static.igem.org/mediawiki/2011/8/83/Urea_modeling.png" alt="fig7" width="750px" />
 +
<div style="font-size:larger">
 +
<b>2NH<sub>3</sub> + HCO<sub>3</sub><sup>-</sup> + 3ATP + H<sub>2</sub>O + NADPH + NAD<sup>+</sup>  
 +
&rarr; Urea + 2ADP + AMP + 2Pi + PPi + NADP<sup>+</sup> + NADH</b>
 +
</div>
 +
Fig. 7 One of the urea producing cycles leaded by the concept of elementary flux modes<br />
 +
*The numbers indicate the relative flux carried by the enzymes.
 +
</div>
 +
 +
<p>
 +
As shown in Fig. 7, we deduced that the carbon atom of urea is provided from HCO<sub>3</sub><sup>-</sup> , which is a byproduct of respiration and therefore is already an abundant compound in the bacterial cytoplasm. On the other hand, we also confirmed that carbamoyl phosphate is a nitrogen source for urea production.We also found that the function of L-glutamine in the urea cycle is to provide nitrogen for urea production via carbamoyl phosphate, because ammonium ion can restore L-glutamine from L-glutamate (which is a
 +
byproduct of the reaction that yields carbamoyl phosphate as a product).This conclusion was confirmed experimentally by Mendel <i>et al</i>. (1996). Also, since only providing a nitrogen source is enough to increase urea production by <i>E. coli</i>, we can also conclude that the aritificial urea cycle in <i>E. coli</i> is stoichiometrically well designed. By comparing Fig. 5 and Fig. 7 we can also observe that, in Fig. 7, the reaction which converts L-glutamate to L-ornithine is not needed for urea production.
 +
</p>
 +
 
 +
<h4 id="production">3.3.2 Finding Modes to Increase the Urea production by <i>E. coli</i></h4>
 +
<p>
 +
There are two ways to obtain more products from a cycle of reactions: increasing the speed the reactions and increasing the concentration of the reactants. This becomes obvious if we think of the cycle as a track which is travelled by cars (the reactants), and the products as the total sum of the number of laps made by every car. If we double the speed of the cars the number of laps will also double (Fig. 8, lower left). Similarly, if we double the number of cars the number of laps will double as well (Fig. 8, lower right). We applied this analogy to the urea cycle, where the metabolites in the cycle are represented by the cars and the total number of laps represents the total urea yield (as shown in the figure below).<br />
 +
Increasing the velocity of the cars corresponds to increasing the amount of carbamoyl phosphate in the urea cycle, because the reaction which converts L-glutamine to carbamoyl phosphate is the rate-limiting reaction of the cycle. On the other hand, increasing the number of the cars correspond to increasing the concentration of the compounds of the urea cycle. We focused on increasing the concentration the compounds of the urea cycle to find ways to increase the urea yield.
 +
</p>
 +
<div class="graph_title">
 +
<img src="https://static.igem.org/mediawiki/2011/d/d6/Cars.png" alt=T(0) width="700px" /><br />
 +
<p>Fig. 8 Two ways to increase urea production</p>
 +
</div>
 +
<p>
 +
L-ornithine, L-citrulline, N-(L-arginino)succinate and L-arginine are four important compounds of the urea cycle. As can be seen I Fig.7, these compounds form a sub-cycle that directly yields urea. Therefore, by increasing the yield of this cycle we can increase the production of urea in <i>E. coli</i>.
 +
</p>
 +
 
 +
<p>
 +
We determined the elementary modes which produce these four important compounds.
 +
All elementary flux modes which produce these compounds from L-glutamine or from compounds in TCA cycle produce L-ornithine as intermediate or final product (these modes are shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/figures#Elem2">Fig. 9</a> and each reaction formula is shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table5">Table 5</a>, it can be concluded that increasing the concentration of L-ornithine will increase the production of urea. One of the L-ornithine producing modes is shown in Fig. 10.
 +
</p>
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2011/8/89/Urea-fig11.png" alt="fig11" width="600px" />
 +
</center>
 +
<div class="graph_title">
 +
<div style="font-size:larger">
 +
<small><b>2-oxoglutarate + NH<sub>3</sub> + acetyl-CoA + ATP + 3NADPH + 3H<sup>+</sup>
 +
&rarr; L-ornithine + CoASH + acetate + ADP + Pi + H<sub>2</sub>O + 3NADP<sup>+</sup></b></small>
</div>
</div>
-
+
Fig. 10 One of the L-ornithine producing pathways from intermediates of TCA cycle<br />
-
MG1655 and JD24293 were transformed separately with pSB3K3,
+
*The numbers indicate the relative flux carried by the enzymes.
-
pTrc-rocF or pTRC-rocF-Arg box. A detailed method is described here.
+
</div>
-
 
+
 
-
Urea concentrations detected in growth media of bacterial samples 1 hour
+
<p>
-
after IPTG induction are shown in Fig.5.Detialed procedure is described here
+
The reactions we determined increase the above mentioned four compounds of the urea cycle are shown in Fig. 9. All modes include the reaction that yields L-ornithine by converting L-glutamate to L-ornithine. <br />
-
+
We also confirmed that <span class="name">E. coli</span> has no feasible routes for production of these four components other than those indicated in Fig. 5. Therefore, we can conclude that the reaction which converts L-glutamate to L-ornithine is a key reaction to increase the reaction rates in the urea cycle and thereby to increase urea production. It should be noted that one of the reactions of the cycle shown in Fig. 5 (the one in the lowest part of the image) requires ATP, NADPH, Acetyl-CoA, and L-glutamate. With the exception of L-glutamate, all of these compounds are already abundant in the cell. Therefore, in future wet experiments, we will focus on studying the effects of supplying L-glutamate to <i>E. coli</i>. We will confirm that by supplying L-glutamate the concentration of intermediates like L-ornithine can be increased and therefore urea production can be increased.
-
<img src="https://static.igem.org/mediawiki/2011/c/cb/TokyoTech_urea-assay1.png" alt="fig4 Assay data"/>
+
</p>
-
<div class="graph_title">
+
<p>
-
Fig.4 Urea concentration in growth media 1 hour after IPTG induction
+
Furthermore, to supply L-glutamine, L-glutamate and L-arginine is effective way to increase the amount of ornithine.(Fig. 11)
-
</div>
+
</p>
-
+
<div align="center">
-
In MG1655(ArgR+), addition of Trc promoter-rocF led to more production of
+
<img src="https://static.igem.org/mediawiki/2011/a/a6/Urea_modeling_overview.png" alt="fig.11a" width="600px" /><br />
-
urea compared to the bare backbone pSB3K3 as expected. These results
+
<span class="graph_title">Fig. 11 Ornithine is made from L-glutamine, L-glutamate and L-arginine</span>
-
show that insertion of rocF resulted in arginase production as expected,  
+
</div>
-
therefore completing the urea cycle in E.coli. In the same strain, however,
+
<p>
-
addition of Arg box sequence led to little change in urea production.
+
We also noted that since L-aspartate is consumed in protein biosynthesis, this amino acid should be supplied from in the medium or produced by <i>E. coli</i> itself not only for increasing the amount of urea production, but also for maintaining the cycle.
-
The reason why the effect of Arg boxes was not apparent is probably that
+
-
pSB3K3 is a low-copy-number plasmid, in contrast to high-copy number used
+
-
in the previous report. A low-copy-number plasmid is not capable of
+
-
introducing enough number of Arg boxes to effectively deactivate the
+
-
arginine repressor. Both of the plasmids containing rocF gene in the
+
-
stain JD24293(Arg-) produce urea more efficiently than those in MG1655.
+
-
+
-
These results are in line with the fact that JD24293 carries argR
+
-
(a gene which codes arginine repressor) loss-of-function mutant, which
+
-
means deactivation of arginine repressor by Arg boxes is not needed and
+
-
addition of the Arg box does not result in a significant increase of urea production.
+
</p>
</p>
 +
 +
<p>
 +
In conclusion, increasing the concentration of L-glutamine, L-glutamate, L-arginine and L-aspartate is an effective way to increase the amount of urea produced.
 +
</p>
 +
 +
 +
<h3 id="future">3.4 Future Work</h3>
 +
<p>
 +
As a future work, we will experimentally confirm our results to show that activating the reactions which supply these amino acids is an effective way to increase the production of urea by <i>E. coli</i>.
 +
</p>
 +
 +
<h3 id="reference">Reference</h3>
 +
[1] Stefan Schuster, <i>et al.</i> A general definition of metabolic pathways useful for systematic organization and analysis of complex metabolic network, Nat Biotechnol(2000) 18:326-32<br />
 +
[2] Mendel Tuchman, <i>et al.</i> Enhanced production of arginine and urea by genetically engineered Escherichia coli K-12 strains, Apple Environ Microbiol(1997) 63: 38-8<br />
 +
 +
<!-- ############ End of main contents ############ -->
<!-- ############ End of main contents ############ -->
Line 471: Line 643:
</p>
</p>
<!-- end of under contents -->
<!-- end of under contents -->
 +
<div style="bottom: 0;left: 30; position:fixed; background-color: #000000; opacity:0.8;">
 +
<a onclick="scrollTo(0,0);return false;" onfocus="this.blur();" style="color:#FFFFFF;">Return to Page Top</a>
 +
</div>
<!-- DO NOT WRITE UNDER HERE -->
<!-- DO NOT WRITE UNDER HERE -->
</body>
</body>
</html>
</html>

Latest revision as of 02:57, 29 October 2011

Tokyo Tech 2011

Urea cooler

1. Abstract

Coolers can be made by dissolving urea in water because it is an endothermic reaction. So, in order to obtain urea, we made the urea cycle in E.coli by introducing rocF gene encoding arginase, which is an enzyme that converts L-arginine to L-ornithine and urea. But just by introducing rocF, only a little amount of urea can be produced because arginine biosynthesis is repressed by the arginine repressor. Therefore, we tried to derepress arginine biosynthesis in two ways. One is by introducing arginine operator sequences(Arg boxes), which bind the arginine repressor. The other is by using a strain that carries a mutation in a gene which encodes arginine repressor. Furthermore, we studied elementary flux modes to provide more urea. As a result, we found out that the artificial urea production system is robust in a stoichiometric point of view. The analysis also revealed that supplementation of arginine, glutamic acid and aspartic acid would increase urea production rate.

Assay data Fig5
Fig. 3 Urea concentration in growth media 1 hour after IPTG induction Fig. 5 The reactions related with the urea cycle

2. Genetic Engineering for Urea Production

2.1 Introduction

Coolers can be made by dissolving urea in water, since dissolution of urea in water is an endothermic reaction (-57.8 cal/g). So we came up with an idea of creating E. coli that can synthesize urea. Originally, E. coli has all the enzymes in the urea cycle except for arginase, which converts L-arginine into L-ornithine and urea(Fig. 1). So we attempted to complete the urea cycle in E. coli by introducing a gene which encodes arginase and obtain urea.
In this work, introduction of the Bacillus subtilis rocF gene which encodes arginase on a standardized plasmid completed urea cycle and enabled E.coli to produce urea as reported by TUCHMAN et al.,(1997).

Urea cycle; Fig1
Fig. 1 Addition of a gene which encoding arginase completes urea cycle in E.coli.

However, just by introducing arginase, E. coli produces only a little amount of urea. TUCHMAN et al. proposed that catabolite repression in arginine biosynthesis pathway is the main reason for the low efficiency of production(TUCHMAN et al., 1997). The bacterial arginine biosynthetic genes are all regulated via a common repressor protein (arginine repressor) encoded by the argR gene. The arginine represor is activated in the presence of arginine (Fig. 2).
TUCHMAN et al. circumvented the repression by introduction of arginine operator sequences (Arg boxes), which bind the arginine repressor. With the arginine repressor binding to Arg boxes, the amount of the arginine repressor which can repress arginine biosynthesis is reduced. In this work, we tried two ways of derepressing arginine biosynthesis. One way is by introducing the Arg boxes as previous work. The other way is by using an E. coli strain that has an argR deletion genotype so that the repressor is not synthetized.

Arginine biosynthesis is repressed by arginine repressor in the presence of arginine.
Fig. 2 Arginine biosynthesis is repressed by the arginine repressor in the presence of arginine.

2.2 Charcterization of rocF and Arg box

Introduction of rocF gene on our part BBa_K649301 led to production of more urea compared to negative controls. Therefore, we confirmed that insertion of rocF gene resulted in arginase production and completed the urea cycle in E. coli as expected.

Fig. 3 Urea concentration in growth media when rocF or Arg box were introduced strain MG1655.

As a way to derepress arginine biosynthesis we introduced Arg box on our part BBa_K649401 in addition to rocF gene (BBa_K649301). This led to production of even more urea. These results show that Arg boxes are effectively derepressing arginine production by deactivating the arginine repressor. Detailed information is shown here.

2.3 Characterization of Ptrc-RBS-rocF-Arg box

In this study Arg boxes and rocF were introduced separately on high-copy-number plasmids (pSB1C3) and low-copy-number plasmids (pSB3K3). We also tested the effect of Arg boxes when they were introduced downstream of rocF gene on low-copy-number plasmids (pSB3K3 and pSB6A1). Here, Arg boxes didn’t work effectively. This is probably because a low-copy-number plasmid is not capable of introducing enough number of Arg boxes to effectively deactivate the arginine repressor. Detailed information is shown here.

2.4 Use of mutation in argR gene

Fig. 4 Urea concentration in growth media when rocF gene on pSB3K3 was introduced in strain MG1655 (argR +) or JE6852 (argR -).

As another way to derepress arginine biosynthesis, we used an E. coli strain that has an argR deletion genotype (JE6852, This strain was obtained from National Institute of Genetics). In our assay results, much more urea was produced when rocF gene was introduced in JE6852 than in MG1655 as expected. See more details about this experiment.

3. Flux analysis for providing more urea

3.1 Abstract

This section is about a metabolic engineering study we did about the urea cycle. On the first part we show how we used “elementary flux modes” (Schuster et al., 2000) to analyze the function of the compounds involved in the urea cycle. Mainly we deduced which compounds act as sources of carbon and sources of nitrogen for the production of urea. On the second part of this study we show how we determined elementary flux modes of the urea cycle to find ways to increase the yield of urea. We focused on a strategy which involves increasing the concentration of four components of the cycle and which we concluded would yield more urea. To confirm our results future experiments will be done.

Fig5
Fig. 5 The reactions related with the urea cycle

*The orange letters are the abbreviated names of the enzymes involved. The red letters are the enzyme expressed by introducing rocF gene. For complete names of the enzymes see Table 3.

3.2 Introduction

3.2.1 What is Elementary Flux Modes?

In Metabolic Engineering, mathematical modeling is an effective way to increase the products of a reaction. In particular, Flux Analysis, which is based on the hypothesis that the system is in a steady state, is effective to find how to increase these products. The concept of elementary flux mode provides a mathematical tool to define and comprehensively describe all metabolic routes that are both stoichiometrically and thermodynamically feasible for a group of enzymes. As a method of metabolic flux analysis, it is based on the hypothesis that the concentration of the reactants and products involved in the cycle does not change.

3.2.2 Analyzing the function of the compounds involved in the Urea Cycle by determining the elementary flux modes

By determining the elementary flux modes of a cycle we can have a more clear view of the function of each of the compounds involved in the cycle being analyzed. Based on the elementary flux modes of the urea cycle, in this study we could deduce that HCO3- acts as the source of carbon for urea production and that both L-glutamine and NH3 act as nitrogen sources for the formation of urea.

T(0) T9 Fig7

3.2.3 Finding Modes to Increase the Urea production by E. coli

In this study we determined elementary flux modes to maximize urea production by E. coli. We found that there are two main strategies to increase urea production: one is to increase the amount of carbamoyl phosphate (which formation is known to be the rate-limiting step of the urea cycle). The other one is to increase the concentration of four components of the urea cycle: L-ornithine, L-citrulline, N-(L-arginino)succinate and L-arginine. We deduced the latter strategy by determining the elementary modes of the urea cycle, and therefore in this study we will focus on the description of this strategy.

T(0)
Fig. 8 Two ways to increase urea production

3.3 Results

In our study, we considered the enzymatic reactions shown in Table 3 to determine the elementary flux modes related to urea production by E. coli. The scheme the overall reaction system is shown in Fig. 5 below.

Fig5
Fig. 5 The reactions related with the urea cycle

*The orange letters are the abbreviated names of the enzymes involved. The red letters are the enzyme expressed by introducing rocF gene. For complete names of the enzymes see Table 3.

By determining the elementary flux modes to produce urea inside E. coli, we found two important results:

  1. We confirmed both L-glutamine and NH3 act as nitrogen providers in the urea cycle, as well as deducing that HCO3- acts as the source of carbon for urea production. These modes did not make use of organic intermediates. Even though L-glutamine is consumed in order to to transfer the side-chain ammonium group needed for the production of carbamoyl phosphate (which in turn transfers the ammonium group to the urea cycle), free ammonium ion can restore L-glutamine from L-glutamate (which is a byproduct of the reaction that yields carbamoyl phosphate as a product).

  2. We concluded that increasing the concentration of L-ornithine will increase the concentration of three related compounds (L-citrulline, N-(L-arginino)succinate, and L-arginine) and this will ultimately lead to an increase in the production of urea. We also noted that since the L-aspartate amino acid, which is needed in the urea cycle we considered(Fig. 5), is normally consumed in protein biosynthesis, so it should be supplied in the culture medium or synthetized by E. coli in order to be able to increase the amount of urea and to maintain the cycles that produce it.

Below is a detailed description of these three results.

3.3.1 Analyzing the function of the compounds involved in the Urea Cycle by determining the elementary flux modes

The first step was to determine the flux modes which need of L-glutamine as an input (Mendel et al., 1996). We did this by calculations based on a matrix as the tableau shown below.

T(0)
T9

Details about the calculations can be found here

We found eight modes that can produce urea without using organic intermediates. These are shown in Fig. 6. Each reaction formula is shown in Table 4. In particular, we focused on one the mode displayed in Fig. 7.

fig7
2NH3 + HCO3- + 3ATP + H2O + NADPH + NAD+ → Urea + 2ADP + AMP + 2Pi + PPi + NADP+ + NADH
Fig. 7 One of the urea producing cycles leaded by the concept of elementary flux modes
*The numbers indicate the relative flux carried by the enzymes.

As shown in Fig. 7, we deduced that the carbon atom of urea is provided from HCO3- , which is a byproduct of respiration and therefore is already an abundant compound in the bacterial cytoplasm. On the other hand, we also confirmed that carbamoyl phosphate is a nitrogen source for urea production.We also found that the function of L-glutamine in the urea cycle is to provide nitrogen for urea production via carbamoyl phosphate, because ammonium ion can restore L-glutamine from L-glutamate (which is a byproduct of the reaction that yields carbamoyl phosphate as a product).This conclusion was confirmed experimentally by Mendel et al. (1996). Also, since only providing a nitrogen source is enough to increase urea production by E. coli, we can also conclude that the aritificial urea cycle in E. coli is stoichiometrically well designed. By comparing Fig. 5 and Fig. 7 we can also observe that, in Fig. 7, the reaction which converts L-glutamate to L-ornithine is not needed for urea production.

3.3.2 Finding Modes to Increase the Urea production by E. coli

There are two ways to obtain more products from a cycle of reactions: increasing the speed the reactions and increasing the concentration of the reactants. This becomes obvious if we think of the cycle as a track which is travelled by cars (the reactants), and the products as the total sum of the number of laps made by every car. If we double the speed of the cars the number of laps will also double (Fig. 8, lower left). Similarly, if we double the number of cars the number of laps will double as well (Fig. 8, lower right). We applied this analogy to the urea cycle, where the metabolites in the cycle are represented by the cars and the total number of laps represents the total urea yield (as shown in the figure below).
Increasing the velocity of the cars corresponds to increasing the amount of carbamoyl phosphate in the urea cycle, because the reaction which converts L-glutamine to carbamoyl phosphate is the rate-limiting reaction of the cycle. On the other hand, increasing the number of the cars correspond to increasing the concentration of the compounds of the urea cycle. We focused on increasing the concentration the compounds of the urea cycle to find ways to increase the urea yield.

T(0)

Fig. 8 Two ways to increase urea production

L-ornithine, L-citrulline, N-(L-arginino)succinate and L-arginine are four important compounds of the urea cycle. As can be seen I Fig.7, these compounds form a sub-cycle that directly yields urea. Therefore, by increasing the yield of this cycle we can increase the production of urea in E. coli.

We determined the elementary modes which produce these four important compounds. All elementary flux modes which produce these compounds from L-glutamine or from compounds in TCA cycle produce L-ornithine as intermediate or final product (these modes are shown in Fig. 9 and each reaction formula is shown in Table 5, it can be concluded that increasing the concentration of L-ornithine will increase the production of urea. One of the L-ornithine producing modes is shown in Fig. 10.

fig11
2-oxoglutarate + NH3 + acetyl-CoA + ATP + 3NADPH + 3H+ → L-ornithine + CoASH + acetate + ADP + Pi + H2O + 3NADP+
Fig. 10 One of the L-ornithine producing pathways from intermediates of TCA cycle
*The numbers indicate the relative flux carried by the enzymes.

The reactions we determined increase the above mentioned four compounds of the urea cycle are shown in Fig. 9. All modes include the reaction that yields L-ornithine by converting L-glutamate to L-ornithine.
We also confirmed that E. coli has no feasible routes for production of these four components other than those indicated in Fig. 5. Therefore, we can conclude that the reaction which converts L-glutamate to L-ornithine is a key reaction to increase the reaction rates in the urea cycle and thereby to increase urea production. It should be noted that one of the reactions of the cycle shown in Fig. 5 (the one in the lowest part of the image) requires ATP, NADPH, Acetyl-CoA, and L-glutamate. With the exception of L-glutamate, all of these compounds are already abundant in the cell. Therefore, in future wet experiments, we will focus on studying the effects of supplying L-glutamate to E. coli. We will confirm that by supplying L-glutamate the concentration of intermediates like L-ornithine can be increased and therefore urea production can be increased.

Furthermore, to supply L-glutamine, L-glutamate and L-arginine is effective way to increase the amount of ornithine.(Fig. 11)

fig.11a
Fig. 11 Ornithine is made from L-glutamine, L-glutamate and L-arginine

We also noted that since L-aspartate is consumed in protein biosynthesis, this amino acid should be supplied from in the medium or produced by E. coli itself not only for increasing the amount of urea production, but also for maintaining the cycle.

In conclusion, increasing the concentration of L-glutamine, L-glutamate, L-arginine and L-aspartate is an effective way to increase the amount of urea produced.

3.4 Future Work

As a future work, we will experimentally confirm our results to show that activating the reactions which supply these amino acids is an effective way to increase the production of urea by E. coli.

Reference

[1] Stefan Schuster, et al. A general definition of metabolic pathways useful for systematic organization and analysis of complex metabolic network, Nat Biotechnol(2000) 18:326-32
[2] Mendel Tuchman, et al. Enhanced production of arginine and urea by genetically engineered Escherichia coli K-12 strains, Apple Environ Microbiol(1997) 63: 38-8

Return to Page Top