Team:Hong Kong HKUST/Project/module4

From 2013.igem.org

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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, isocitrate lyase and malate synthase, in glyoxylate cycle. These two enzymes are encoded by <i>aceA</i> and <i>aceB</i> genes, respectively. 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, mitochondrial leader sequence, tag protein and terminator that will allow the expression of prokaryotic gene in eukaryotic cell. In addition to the constitutive system, we assemble an 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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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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Revision as of 14:14, 27 September 2013


Glyoxylate Shunt

Overview

Our artificial futile cycle is driven by the expression of two key enzymes of glyoxylate cycle, isocitrate lyase (aceA) and malate synthase (aceB). Since mammalian cells lacks genes expressing glyoxylate shunt, we introduce these genes form E. coli 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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