Team:Manchester/fattytest

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<div class="global">
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             <p><b><a id="Q1">Introduction</a></b><br>
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            <p> <b>Introduction</b> <br>
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Since the model of the fatty acid biosynthesis pathways highlighted several key enzymes that could be altered to produce palm oil, including Delta9, Delta12 and FabA, which agrees with previous publications on these enzymes. Delta9 and Delta12 are involved in linear pathways, meaning that there are obvious reactants and products, so the overexpression of them can be measured directly via LC-MS and other techniques. However, FabA is a β-Hydroxydecanoyl Thiol Ester Dehydrase involved in a cyclic pathway [1] specifically the conversion of β-hydroxy acyl-ACP to Enol acyl-ACP as part of the fatty acid biosynthetic pathway [2].
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Since the model of the fatty acid biosynthesis pathways highlighted several key enzymes that could be altered to produce palm oil, including Delta9, Delta12 and FabA, which agrees with previous publications on these enzymes. Delta9 and Delta12 are involved in linear pathways, meaning that there are obvious reactants and products, so the overexpression of them can be measured directly via LC-MS and other techniques. However, FabA is a β-Hydroxydecanoyl Thiol Ester Dehydrase involved in a cyclic pathway [1] specifically the conversion of β-hydroxy acyl-ACP to Enol acyl-ACP as part of the fatty acid biosynthetic pathway [2]. Therefore characterising any specific change due to FabA overexpression will be challenging, as any products will automatically be involved in the proceeding stage of the cycle and it would therefore be difficult to determine the effect of overexpression. An alternative to measure the overexpression of FabA, would be through the addition of His-tags to either the N-terminal and or the C-terminal of FabA. However, depending on the structure of FabA, the addition of His-tags could potentially interfere with expression, protein folding, enzymatic functions and interactions [3, 4, 5, 6 and 7]. Several studies including work by [5] and [6], respectively showed that the addition of C-Terminus His-tags to proteins could interfere with enzyme activity and alter di-sulphide and therefore protein structure. These problems are particularly applicable to FabA, as it forms a homodimer, as shown by Leesong et al., 1996 [1]. Therefore, the addition of His-tag could potentially interfere with the interaction domain and thus the formation of a homodimer, which would be consistent with several reports [4, 5 and 6].  To address this issue, we decided to perform a molecular dynamics simulation using the GROMACS software package [8] on a structure of FabA, determined by X-ray crystallography by Leesong et al., 1996 [1]. This would allow for the trajectories of the N- and C-Termini of FabA over the course of the simulation to be studied, therefore allowing us to identify which terminal would be more suited for His-tag addition.
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                   <div class="block3">
                     <a href="https://2013.igem.org/Team:Manchester/parametertest">PARAMETER ESTIMATION</a>
                     <a href="https://2013.igem.org/Team:Manchester/parametertest">PARAMETER ESTIMATION</a>
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                    <a href="https://2013.igem.org/Team:Manchester/fabAmodeltest">fabA PROTEIN MODEL</a>
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                    <a href="https://2013.igem.org/Team:Manchester/popdynamictest">POPULATION DYNAMICS</a>
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                   <div class="block4">
                     <a href="https://2013.igem.org/Team:Manchester/collabtest">MODELLING COLLABORATION</a>
                     <a href="https://2013.igem.org/Team:Manchester/collabtest">MODELLING COLLABORATION</a>
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                  <div class="question1">
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                    <a href="#Q1">Introduction</a>
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                    <a href="#Q2">Installing GROMACS</a>
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                    <a href="#Q3">Behind the scenes of a Molecular Dynamics Simulation</a>
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                    <a href="#Q4">Getting a working GROMACS simulation for FabA</a>
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                    <a href="#Q5">To His-tag or not to His-tag</a>
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Latest revision as of 14:48, 29 September 2013

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Top

Safety

Introduction
Since the model of the fatty acid biosynthesis pathways highlighted several key enzymes that could be altered to produce palm oil, including Delta9, Delta12 and FabA, which agrees with previous publications on these enzymes. Delta9 and Delta12 are involved in linear pathways, meaning that there are obvious reactants and products, so the overexpression of them can be measured directly via LC-MS and other techniques. However, FabA is a β-Hydroxydecanoyl Thiol Ester Dehydrase involved in a cyclic pathway [1] specifically the conversion of β-hydroxy acyl-ACP to Enol acyl-ACP as part of the fatty acid biosynthetic pathway [2].

MODELLING
FATTY ACID PRODUCTION
PARAMETER ESTIMATION
fabA PROTEIN MODEL
POPULATION DYNAMICS
MODELLING COLLABORATION

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