Team:Hong Kong HKUST/Project/module4

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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Wetlab">Wetlab</a>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Wetlab">Wetlab</a>
<ul>
<ul>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/experiment">Experiments</a></li>
 
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook">Notebook</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook">Notebook</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/protocols">Protocols</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/protocols">Protocols</a></li>
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<h6>Glyoxylate Shunt</h6>
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<h6>Modules</h6>
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<a href=#1>Overview</a>
 
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<a href=#3>Protein Expression</a>
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<a href=#4>Characterization</a>
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Glyoxylate Shunt
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<ul><li>
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<a href=#1>Overview</a>
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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Project/module1">FA Quantification & Cell Viability</a>
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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Project/module2">FA Sensing Mechanism</a>
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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Project/module4">Protein Trafficking</a>
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                                      <a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Project/module3">Protein Trafficking</a>
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Glyoxylate Shunt
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<h3>Overview</h3>
<h3>Overview</h3>
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Our artificial futile cycle is driven by the expression of two key enzymes of glyoxylate cycle, isocitrate lyase (<i>aceA</i>) and malate synthase (<i>aceB</i>). Since mammalian cells lacks genes expressing glyoxylate shunt,  we introduce these genes form <i>E. coli</i> and assemble them in a constitutive construct composed of mammalian constitutive promoter (CMV Promoter and EF-1alpha Promoter respectively), mitochondrial leader sequence, and hGH terminator that will allow the expression of prokaryotic gene in eukaryotic cell. In addition to the constitutive system, we assemble a fatty acid inducible construct which will allow tunable gene expression according to the concentration of fatty acid in the medium. We decided to introduce inducible system to prevent fatty acid deficiency in low concentration of plasma fatty acid and facilitate greater fatty acid uptake at a high circulating fatty acid levels. The inducible and constitutive system will be compared in terms of fatty acid uptake rate in a range concentration of fatty acid. 
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<p>Our artificial futile cycle design is based on the tested findings by Dean et  al, who demonstrated that by introducing the artificial glyoxylate shunt in mouse  liver cells, fatty acid uptake would increase and the mice would acquire  resistance against obesity when fed with fatty diet. (Dean, 2009) In essence,  we are reproducing their work from scratch but through the use of standard BioBricks.</p>
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<center><img src="https://static.igem.org/mediawiki/2013/a/a7/Glyoxylate_pathway.jpg" style="width:100%"></center>
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Dean, Jason T. Resistance to Diet-Induced Obesity in Mice with Synthetic Glyoxylate Shunt. 2009. Graphic.
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<br><br>
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<p>In Dean et al.&rsquo;s  work, the glyoxylate shunt was achieved by the expression of two key enzymes  from the bacterial glyoxylate cycle, isocitrate lyase (AceA) and malate synthase (AceB). When the two enzymes enter mitochondria in liver cells, isocitrate  lyase will convert a proportion of isocitrate into glyoxylate, which will then  be converted by malate synthase into malate. This process would bypass the pathway  through alpha-ketoglutarate, and therefore, bypassing the ATPs and reducing  equivalent generating steps. (Dean, 2009)</p>
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<br>
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<p>To reproduce  this masterpiece, we would first need to convert every single part into  BioBricks: we cloned out the glyoxylate enzymes genes <em>aceA</em> and <em>aceB</em> from <em>E.  coli</em> and assembled them with mitochondrial leader sequence (MLS). The two  translation units were then assembled downstream of mammalian constitutive CMV  Promoter and EF-1alpha Promoter respectively. Lastly, the hGH polyA signal  sequence was added to serve as terminator of the construct. These constructs,  when put together, should return the original constitutive glyoxylate shunt.</p>
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<br>
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<p>Yet, in  addition to the constitutive system, we are assembling a fatty acid inducible  construct that allows tunable gene expression according to the concentration of  fatty acid around. We decided to introduce this inducible system to prevent  fatty acid deficiency when the concentration of fatty acid in body is low,  hopefully overcoming the foreseeable shortcomings of the original constitutive shunt.</p><br>
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<p>Lastly, the  inducible and constitutive system will be compared in terms of fatty acid  uptake rate in a range concentration of fatty acid and their performances shall  be evaluated.</p>
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<h3>Reference</h3>
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<p>Dean Jason T, Tran Linh et al. "Resistance to Diet-Induced Obesity in Mice with Synthetic Glyoxylate Shunt." (2009)</p>
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<div class="nine columns"><p id="2"></p>
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Latest revision as of 12:42, 28 October 2013


Glyoxylate Shunt

Overview

Our artificial futile cycle design is based on the tested findings by Dean et al, who demonstrated that by introducing the artificial glyoxylate shunt in mouse liver cells, fatty acid uptake would increase and the mice would acquire resistance against obesity when fed with fatty diet. (Dean, 2009) In essence, we are reproducing their work from scratch but through the use of standard BioBricks.


Dean, Jason T. Resistance to Diet-Induced Obesity in Mice with Synthetic Glyoxylate Shunt. 2009. Graphic.

In Dean et al.’s work, the glyoxylate shunt was achieved by the expression of two key enzymes from the bacterial glyoxylate cycle, isocitrate lyase (AceA) and malate synthase (AceB). When the two enzymes enter mitochondria in liver cells, isocitrate lyase will convert a proportion of isocitrate into glyoxylate, which will then be converted by malate synthase into malate. This process would bypass the pathway through alpha-ketoglutarate, and therefore, bypassing the ATPs and reducing equivalent generating steps. (Dean, 2009)


To reproduce this masterpiece, we would first need to convert every single part into BioBricks: we cloned out the glyoxylate enzymes genes aceA and aceB from E. coli and assembled them with mitochondrial leader sequence (MLS). The two translation units were then assembled downstream of mammalian constitutive CMV Promoter and EF-1alpha Promoter respectively. Lastly, the hGH polyA signal sequence was added to serve as terminator of the construct. These constructs, when put together, should return the original constitutive glyoxylate shunt.


Yet, in addition to the constitutive system, we are assembling a fatty acid inducible construct that allows tunable gene expression according to the concentration of fatty acid around. We decided to introduce this inducible system to prevent fatty acid deficiency when the concentration of fatty acid in body is low, hopefully overcoming the foreseeable shortcomings of the original constitutive shunt.


Lastly, the inducible and constitutive system will be compared in terms of fatty acid uptake rate in a range concentration of fatty acid and their performances shall be evaluated.

Reference

Dean Jason T, Tran Linh et al. "Resistance to Diet-Induced Obesity in Mice with Synthetic Glyoxylate Shunt." (2009)

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