Team:Manchester/Parts

From 2013.igem.org

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             <!--<p id="footer"><b>Figure 1. Two colour image of FabA homodimer with labelled N- and C-Termini (arrows)</b><br>-->
             <!--<p id="footer"><b>Figure 1. Two colour image of FabA homodimer with labelled N- and C-Termini (arrows)</b><br>-->
             <p><b><a id="Q1">Introduction</a></b><br>
             <p><b><a id="Q1">Introduction</a></b><br>
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Our model of the fatty acid biosynthesis pathways highlighted several key enzymes that could be altered to produce palm oil, including Delta 9, Delta 12 and FabA, which agrees with previous publications on these enzymes. Delta 9 and Delta 12 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<sup>[1]</sup> specifically the conversion of β-hydroxy acyl-ACP to Enol acyl-ACP as part of the fatty acid biosynthetic pathway<sup>[2]</sup>. 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<sup>[3][4][5][6][7]</sup>. Several studies including work by <sup>[5]</sup> and <sup>[6]</sup>, 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<sup>[1]</sup>. 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<sup>[4][5][6]</sup>. To address this issue, we decided to perform a molecular dynamics simulation using the GROMACS software package<sup>[8]</sup> on a structure of FabA, determined by X-ray crystallography by Leesong et al., 1996<sup>[1]</sup>. 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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Lorem ipsum dolor sit amet, consectetur adipiscing elit. Mauris vehicula et velit placerat pulvinar. Etiam mollis vel leo laoreet accumsan. Ut pellentesque sollicitudin quam nec gravida. Proin eu tempus augue. Praesent vehicula, lacus quis auctor vulputate, justo nunc lacinia enim, ut imperdiet magna odio at ligula. Phasellus non molestie lorem. Suspendisse semper leo eget est malesuada, ut fringilla nibh posuere. Mauris non enim sit amet odio dictum commodo id iaculis dui. Nam elit tortor, ornare in euismod id, lacinia in eros. Praesent vehicula orci a elit rutrum, in pellentesque nisi tempus.Lorem ipsum dolor sit amet, consectetur adipiscing elit. Mauris vehicula et velit placerat pulvinar. Etiam mollis vel leo laoreet accumsan. Ut pellentesque sollicitudin quam nec gravida. Proin eu tempus augue. Praesent vehicula, lacus quis auctor vulputate, justo nunc lacinia enim, ut imperdiet magna odio at ligula. Phasellus non molestie lorem. Suspendisse semper leo eget est malesuada, ut fringilla nibh posuere. Mauris non enim sit amet odio dictum commodo id iaculis dui. Nam elit tortor, ornare in euismod id, lacinia in eros. Praesent vehicula orci a elit rutrum, in pellentesque nisi tempus.Lorem ipsum dolor sit amet, consectetur adipiscing elit. Mauris vehicula et velit placerat pulvinar. Etiam mollis vel leo laoreet accumsan. Ut pellentesque sollicitudin quam nec gravida. Proin eu tempus augue. Praesent vehicula, lacus quis auctor vulputate, justo nunc lacinia enim, ut imperdiet magna odio at ligula. Phasellus non molestie lorem. Suspendisse semper leo eget est malesuada, ut fringilla nibh posuere. Mauris non enim sit amet odio dictum commodo id iaculis dui. Nam elit tortor, ornare in euismod id, lacinia in eros. Praesent vehicula orci a elit rutrum, in pellentesque nisi tempus.Lorem ipsum dolor sit amet, consectetur adipiscing elit. Mauris vehicula et velit placerat pulvinar. Etiam mollis vel leo laoreet accumsan. Ut pellentesque sollicitudin quam nec gravida. Proin eu tempus augue. Praesent vehicula, lacus quis auctor vulputate, justo nunc lacinia enim, ut imperdiet magna odio at ligula. Phasellus non molestie lorem. Suspendisse semper leo eget est malesuada, ut fringilla nibh posuere. Mauris non enim sit amet odio dictum commodo id iaculis dui. Nam elit tortor, ornare in euismod id, lacinia in eros. Praesent vehicula orci a elit rutrum, in pellentesque nisi tempus.
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           <div class="text1">             
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             <p><b><a id="Q2">Installing GROMACS</a></b><br>
             <p><b><a id="Q2">Installing GROMACS</a></b><br>
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The Groningen Machine for Chemical Simulation (GROMACS) Version 4.5.5 [8], was installed on a MacBook Pro 2011 model, operating OS X 10.8.3, with a 4 GB 1333 MHz DDR3 memory, 2.3 GHz Intel Core i5 processor and a 320 GB SATA disk drive. Prior to installing GROMACS, “command line tools” were installed within Xcode, Version 4.6.1. GROMACS was installed to single precision, with the source file downloaded from www.gromacs.org/Downloads. In addition to GROMACS, the FFTW library, Version 3.3.3<sup>[9]</sup> was installed, with the source file downloaded from www.fftw.org/download.html.
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             <p><b><a id="Q3">Behind the scenes of the Molecular Dynamics Simulations</a></b><br>
             <p><b><a id="Q3">Behind the scenes of the Molecular Dynamics Simulations</a></b><br>
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For the simulations of FabA, a dimeric structure of FabA (PDB ID: 1MKB) derived by X-ray Diffraction, with a resolution of 2 Å from an E.Coli expression system by<sup>[1]</sup> was used.  
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The molecular dynamics simulation was performed within the GROMACS package using version 4.5.5 [8], with the AMBER99SB force field<sup>[10]</sup>, the transferrable intermolecular potential 3P (TIP3P) water model<sup>[11]</sup> and the original crystal waters, from the X-Ray Crystallography with periodic boundary conditions. The methods for generating both topologies and parameters for G16bP are as described above. For all cases of the Protein in water, a protocol
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Revision as of 23:10, 3 October 2013

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Safety

We submitted and sequenced three biobricks integral to our project. These can be found below:

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This year we submitted and characterised 3 BioBricks, one of which is an improvement of BBa_K925000! Characterisation below:

Introduction
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Mauris vehicula et velit placerat pulvinar. Etiam mollis vel leo laoreet accumsan. Ut pellentesque sollicitudin quam nec gravida. Proin eu tempus augue. Praesent vehicula, lacus quis auctor vulputate, justo nunc lacinia enim, ut imperdiet magna odio at ligula. Phasellus non molestie lorem. Suspendisse semper leo eget est malesuada, ut fringilla nibh posuere. Mauris non enim sit amet odio dictum commodo id iaculis dui. Nam elit tortor, ornare in euismod id, lacinia in eros. Praesent vehicula orci a elit rutrum, in pellentesque nisi tempus.Lorem ipsum dolor sit amet, consectetur adipiscing elit. Mauris vehicula et velit placerat pulvinar. Etiam mollis vel leo laoreet accumsan. Ut pellentesque sollicitudin quam nec gravida. Proin eu tempus augue. Praesent vehicula, lacus quis auctor vulputate, justo nunc lacinia enim, ut imperdiet magna odio at ligula. Phasellus non molestie lorem. Suspendisse semper leo eget est malesuada, ut fringilla nibh posuere. Mauris non enim sit amet odio dictum commodo id iaculis dui. Nam elit tortor, ornare in euismod id, lacinia in eros. Praesent vehicula orci a elit rutrum, in pellentesque nisi tempus.Lorem ipsum dolor sit amet, consectetur adipiscing elit. Mauris vehicula et velit placerat pulvinar. Etiam mollis vel leo laoreet accumsan. Ut pellentesque sollicitudin quam nec gravida. Proin eu tempus augue. Praesent vehicula, lacus quis auctor vulputate, justo nunc lacinia enim, ut imperdiet magna odio at ligula. Phasellus non molestie lorem. Suspendisse semper leo eget est malesuada, ut fringilla nibh posuere. Mauris non enim sit amet odio dictum commodo id iaculis dui. Nam elit tortor, ornare in euismod id, lacinia in eros. Praesent vehicula orci a elit rutrum, in pellentesque nisi tempus.Lorem ipsum dolor sit amet, consectetur adipiscing elit. Mauris vehicula et velit placerat pulvinar. Etiam mollis vel leo laoreet accumsan. Ut pellentesque sollicitudin quam nec gravida. Proin eu tempus augue. Praesent vehicula, lacus quis auctor vulputate, justo nunc lacinia enim, ut imperdiet magna odio at ligula. Phasellus non molestie lorem. Suspendisse semper leo eget est malesuada, ut fringilla nibh posuere. Mauris non enim sit amet odio dictum commodo id iaculis dui. Nam elit tortor, ornare in euismod id, lacinia in eros. Praesent vehicula orci a elit rutrum, in pellentesque nisi tempus.

Installing GROMACS
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Behind the scenes of the Molecular Dynamics Simulations
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PROJECT
OVERVIEW
NOTEBOOK
LAB BOOK
PARTS
SAFETY
JUDGING
ATTRIBUTIONS

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