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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<br>
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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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<a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Project/module2">FA Sensing Mechanism</a>
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<h3>Overview</h3>
<h3>Overview</h3>
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Our artificial futile cycle design is based almost entirely 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. (IN TEXT CITATION) In essence, we are reproducing their work from scratch but through the use of standard BioBricks.
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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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[Figure from Dean et al’s paper]
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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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In Dean et al.’s work, the glyoxylate shut 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. (IN TEXT CITATION)
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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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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 shut.
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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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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 shut.
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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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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.
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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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Reference:
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Dean ……..
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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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