Team:Manchester/Parts

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             <p>TITLE</p>
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             <b>This year we submitted and characterised 3 BioBricks, one of which is an improvement of <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K925000" target="_blank">BBa_K925000</a>!</b>
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<center><i>We used an existing BioBrick component with Constitutive promoter and RBS<br><br> (BBa_K608002) to express our BioBrick parts:</i></center><br>
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<p id="footer"><b>Figure 5. Overlay of structures from 1 ns FabA simulation. Images of overlaid from the following respective time points: 0 ps, 250 ps, 500 ps, 750 ps and 1000 ps with the following colours indicating each individual image: Green, Blue, Purple, Orange and Grey, respectively.  Both the N-Terminal and C-Terminal, are specified (Dotted Box), with a zoom in on each respective terminal at an angle appropriate to visualise the positions of the terminals.  
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<!--<p id="footer"><b>Figure 5. Overlay of structures from 1 ns FabA simulation. Images of overlaid from the following respective time points: 0 ps, 250 ps, 500 ps, 750 ps and 1000 ps with the following colours indicating each individual image: Green, Blue, Purple, Orange and Grey, respectively.  Both the N-Terminal and C-Terminal, are specified (Dotted Box), with a zoom in on each respective terminal at an angle appropriate to visualise the positions of the terminals.  
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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>
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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>-->
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             <p><b><a id="Q1">Introduction</a></b><br>
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             <p><b>Successful Expression of delta 9 and delta 12 desaturase, and FabA</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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<p>In order to characterise our biobricks, we made use of the standard parts found in the registry. By inserting the created biobricks BBa_K1027001 and BBa_K1027002 in to BBa_K608002 (a biobrick consisting of a ribosomal binding site and a constitutive promoter), we were able to create a new construct that expressed the delta 9 desaturase, delta 12 desaturase and FabA proteins. The constructs are shown above.</p>
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<p><b>Plates</b></p>
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<p>Another piece of evidence suggesting that our constructs were successfully expressing our delta 9 desaturase and delta 12 desaturase enzymes was the size of the colonies grown on the plates. Pictures of the plates are shown below. On the far left is a control plate, with bacteria transformed with BBa_K608002 but with no gene inserted in front of the promoter. To the right of that are our delta 12 colonies. They are much smaller than the control, and even taking 20 hours to grow to that size as opposed to the usual 16 hours. We hypothesise that because delta 12 desaturase is a membrane-bound protein that the <i>E. coli</i> does not normally express, constitutively expressing it could inhibit the bacteria and slow growth. Compare this to FabA on the far right. Looking through the literature, we found that overexpression of FabA results in no significant difference in growth size or speed relative to the wild type strain (Luo et al, 2009). Compared to the control plate, we also found that the colonies grew normally.</p>
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<p><b>Gel Digests</b></p>
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<p>To confirm that our desired genes were in fact within the expression construct mentioned above, we carried out test digests of our ligated plasmids. Happily, when we ran our digests on an agarose gel, we saw all of the bands we would predict from the expected fragments. The gel pictures are on the right. <br>
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<b>delta 9 desaturase:</b> Cut with BamHI + EcoRV. Expected bands 1424 bp, 1262 bp, 99 bp.<br>
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<b>delta 12 desaturase:</b> Cut with BamHI, XbaI, PstI. Expected bands 2127 bp, 656 bp, 430 bp.<br>
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<b>FabA</b> Cut with EcoRV. Expected bands 1429 bp, 1115 bp. Cut with EcoRV, PstI. Expected bands 1153 bp, 1115 bp, 339 bp.<br></p>
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<p><b>Improvement of existing BioBrick</b></p>
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<p>Our biobrick BBa_K1027002 (<b>delta 12 desaturase</b>) is an improvement of the <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K925000" target="_blank">BBa_K925000</a> biobrick created by the St Andrews team in 2012. We improved this biobrick from the ground up, starting by taking the protein sequence from <i>Synechocystis</i> sp. PCC 6803. This protein sequence was reverse translated and then codon optimised for expression in <i>E. coli</i>. We believe our redesigned biobrick has two features that the original biobrick does not. Firstly, the original biobrick could not be transformed by iGEM HQ, whereas we have managed successful transformation (as shown by the plate pictures). Next, the St. Andrews biobrick was cloned from cyanobacteria, and so is not optimised for expression in <i>E. coli</i>. As St. Andrews then went on to express their delta 12 desaturase biobrick in <i>E. coli</i> it follows that a sequence optimised for expression in this chassis, as ours was, would perform better than the sequence naturally found in cyanobacteria.</p>
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            <p id="footer"><b>LB-Agar (CmR) plates after 20h growth at 37&deg;C. Left: Control plate (transformed with empty vector BBa_K608002). Right: Transformed with delta 12 desaturase (note smaller colonies)</b><br>
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            <p id="footer"><b>LB-Agar (CmR) plates after 20h growth at 37&deg;C. Left: Control plate (transformed with empty vector BBa_K608002). Right: Transformed with FabA gene (note similar sized colonies)</b><br>
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             <p><b><a id="Q2">Installing GROMACS</a></b><br>
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             <p><b>Orbitrap LC-MS analysis</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> Delta 9 desaturase and delta 12 desaturase enzymes were chosen because their products, when expressed in their host organism (<i>Synechocystis</i> sp. PCC 6803), convert stearic acid into <b>oleic acid</b>, and oleic acid into <b>linoleic acid</b> respectively. Therefore, we fed batches of transformed DH5-alpha with 2 different concentrations of exogenous fatty acid (0.1% and 0.5% stearic acid fed to the delta 9 desaturase batch, and 0.1% and 0.5% oleic acid fed to the delta 12 desaturase batch), left the cultures growing overnight and then harvested the cells.<br>
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<br>
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To analyse the metabolites extracted from both wild-type DH5-alpha and DH5-alpha expressing our delta 9 desaturase and delta 12 desaturase enzymes, we made use of the MIB’s in-house Orbitrap Liquid Chromatography - Mass Spectrometry (LC-MS). This technique was chosen because of its high mass accuracy and sensitivity. Upon analysing the most abundant metabolites extracted from our expression strains and comparing this data with the most abundant metabolites extracted from wild-type, it is apparent that a massive increase in linoleic acid (incorporated in phosphatidylethanolamines, PE) has occurred. This is demonstrated in the two figures directly below. The chromatograms produced for the delta 12 desaturase expression strains are also shown below. There is a clear difference between the wild-type <i>E. coli</i> fed with exogenous substrate compared with the <i>E. coli</i> strains expressing delta 12 desaturase. It is probable that the peak appearing around 7.9 min in the delta 12 desaturase strains is due to phospholipid incorporating <b>18:2 (9Z, 12Z) - linoleic acid</b>.
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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>
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            <p id="footer"><b>The four most abundant metabolites extracted from <b>wild-type DH5-alpha</b>. Analysed using Orbitrap LC-MS. Note prominence of short-chain fatty acids. </b><br>
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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>
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            <p id="footer"><b>The four most abundant metabolites extracted from our <b>delta 12 desaturase</b> expression strains. Analysed using Orbitrap LC-MS. Note prominence of C18:2 (9Z, 12Z) (linoleic acid). </b><br>
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            <p id="footer"><b>Figure 2. GROMACS Molecular Dynamics Simulation Workflow. The commands used at each step are included, with a brief description of their function</b><br>
 
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            <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 derived from the “Lysozyme in Water” GROMACS tutorial (by J. Lemkul, Department of Biochemistry, Virginia Tech) and optimized for FabA was used. Briefly, a cubic cell of 2 nm in diameter with the protein centered was used and filled with the generic single point charge 216 (SPC216) water configuration<sup>[12]</sup>. The system was neutralized to a salt concentration of 150 mM by adding Na+ and Cl-. Energy Minimization (EM) was performed using the Steepest Descent Algorithm<sup>[13]</sup> with a tolerance of 1000 KJ mol-1 nm-1, to remove any steric clashes or inappropriate geometries.
 
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In the simulation, the long-range electrostatic interactions were modeled using the Particle-Mesh- Eswald (PME) method <sup>[14][15]</sup> and the Linear Constraint Solver (LINCS) algorithm<sup>[16]</sup> to preserve chemical bond lengths. Temperature and pressure coupling were performed independently, using a modified Berendsen thermostat<sup>[17]</sup> at constant temperature of 300 K at a time constant of 0.1 ps and the Parinello-Rahman barostat algorithm<sup>[18]</sup> at a constant pressure of 1 bar, for a time constant of 2 ps and V-rescale, respectively.
 
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The Molecular Dynamics simulations trajectories were analysed with the GROMACS analysis tools<sup>[19]</sup>, to output structural molecular dynamics trajectories and PyMOL (The PyMOL Molecular Graphics System, Education-Use-Only, Version 1.3 Schrödinger, LLC) used to visualize and create both still structures and videos. All calculations of progression plots from the simulations were produced using the GROMACS analysis tools with output files viewed in the Microsoft Excel 2011.
 
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          <div class="text3">
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        <div class="text4">
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             <p><b><a id="Q4">Getting a working GROMACS simulation for FabA</a></b><br>
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             <img id="left" src="https://static.igem.org/mediawiki/2013/4/40/Orbitrapchromatogramsmanchester.png" width="430" height="450"/>
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To study the motions of the N- and C-Termini of FabA with molecular dynamics, we first had to get an optimized system preparation protocol, which we based on the “Lysozyme in Water” GROMACS tutorial by <sup>[20]</sup>. Firstly, we built a simulation box of 2nm around FabA, which was sufficient to satisfy the minimum image convention and the simulation cut-off schemes without adding excess solvent. FabA had to then be solvated within this box by a solvent configuration compatible with the solvent model applied to the protein, in this case the SPC216<sup>[12]</sup>, which is compatible with the TIP3 water model<sup>[11]</sup>. The system is then neutralized through the addition of ions to a molarity of 150 mM, therefore allowing the system to reach a neutral state. Our results indicate that FabA is well solvated in SPC216 water and neutralized to a molarity of 150 mM in a cubic 2 nm cell.
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<img src="https://static.igem.org/mediawiki/2013/c/c9/Fabafig3.png" width="900" height="258"/>
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<p id="footer"><b>Figure 3. Establishing, solvating and neutralising FabA, for simulation preparation.  A. The establishment of a 2nm simulation cube around the dimerised structure of FabA. B. The solvation of the FabA structure by the addition of SPC216 water molecule as a solvent. C. Neutralisation of the solvated system by the addition of Na+ and Cl- ions. Na+ and Cl- ions are represented by Blue and Green spheres, respectively and SPC216 water is represented by Cyan coloured molecules.</b><p>
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<p>Once we had prepared the system, it was relaxed by energy minimization. This ensures that there are no steric clashes or inappropriate geometries that exist, by applying the steepest descent algorithm<sup>[13]</sup>. With the minimized structure, the system of solvent and ions around the protein are equilibrated to become orientated about the protein solute at the same temperature, by an isothermal-isochoric ensemble<sup>[17]</sup>. Pressure is then applied using an isobaric-isochoric ensemble, thereby ensuring that the system reaches a proper density<sup>[18]</sup>.  Our energy minimized structure shows a decrease from -6.75E+05, to a maximum energy plateau for the system of -1.05 E+06 after 1238 ps of minimization time. The temperature of the system quickly reaches the target value of 300 K remaining stable, with an average temperature of 299.82 K, the equivalent of 27 °C over the 100 ps equilibration. Over the course of the 100 ps equilibration stage, both the pressure and density of the system averages 0 bar and 1015.19 Kg m-3.  This is close to the experimental value of 0 bar and 1000 Kg m-3 and the equivalent of Earth’s atmospheric pressure at sea level and the density of water. The pressure fluctuations are consistent with the applied isothermal-isobaric ensemble and is suggestive of compatible molecular dynamics conditions for simulations of FabA.</p>
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<img src="https://static.igem.org/mediawiki/2013/1/1a/Fabafig4.png" width="900" height="500"/>
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<p id="footer"><b>Figure 4. Graphs of the Energy Minimisation, Temperature, Pressure and Density equilibration of the FabA simulation system prior to simulation. </b><p>
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             <img id="right" src="https://static.igem.org/mediawiki/2013/f/f7/Orbitraplcmsflow.png" width="430" height="450"/>
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             <p><b><a id="Q5">To His-tag or not to His-tag</a></b><br>
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              <p id="footer"><b>Above Left: Chromatograms obtained through Orbitrap LC-MS analysis.<br> Above Right: Workflow of how we went from samples to results<br>Below Left: Bar chart showing increase in linoleic acid when delta 12 desaturase is present<br>Below Right: Heat map showing metabolite abundances </b><br>
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Now that FabA is ready to undergo simulations, we ran a simulation for 1 ns under the notion that we would be able to visualize motions around the N- and C-Terminals during the course of the simulation and therefore determine, which terminal would be more appropriate to add His-tags to.  The conclusion for our simulation was that the N-Terminal is ideal for the addition of His-tags for several reasons. Firstly, the C-terminal is localized in close vicinity to the interaction domain of the FabA homodimer, therefore the addition of His-tags could possibly interfere with the dimer interaction<sup>[6]</sup>. Our model also shows that the C-terminal is more dynamic compared to the N-terminal and there are several times in the simulation that the C-terminal interacts with the dimerization domain and may interfere with the folding and function of the protein<sup>[4][5][6]</sup>. Therefore we concluded that the N-terminal would be ideal to add the His-tags to, as the N-terminal is less dynamic and will be less likely to interfere with folding and the protein function. With this in mind, our experimental team began to design methods to extract FabA and add His-tags to use for the characterization of FabA overexpression.
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<img src="https://static.igem.org/mediawiki/2013/9/9e/Fabafig5.png" width="900" height="640"/>
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<p id="footer"><b>Figure 5. Overlay of structures from 1 ns FabA simulation. Images of overlaid from the following respective time points: 0 ps, 250 ps, 500 ps, 750 ps and 1000 ps with the following colours indicating each individual image: Green, Blue, Purple, Orange and Grey, respectively.  Both the N-Terminal and C-Terminal, are specified (Dotted Box), with a zoom in on each respective terminal at an angle appropriate to visualise the positions of the terminals.
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<iframe src="//player.vimeo.com/video/75638296" width="900" height="300" frameborder="0" webkitallowfullscreen mozallowfullscreen allowfullscreen></iframe>
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            <img id="left" src="https://static.igem.org/mediawiki/2013/3/36/Delta12grapholeicacid.png" width="430" height="450"/>
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            <img id="right" src="https://static.igem.org/mediawiki/2013/0/0d/Orbitrapheatmapmanchester.png" width="430" height=450"/>
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<b>Note</b><br>
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All figures of FabA were produced by us, using the FabA structure (PDB ID: 1MKB found at http://www.rcsb.org/pdb/explore/explore.do?structureId=1MKB) as obtained by Leesong et al 1996
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          <p><b>References</b> <br>
 
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[1]. Leesong, M., Henderson, B.S., Gillig, J.R., Schwad, J.M. and Smith, J.L. 1996. Structure of a dehydratase-isomerase from the bacterial bathways for biosynthesis of unsaturated fatty acids> two catalytic activities in one active site. Structure 4:253-264.<br>
 
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[2]. Xu, P., Gu, G., Wang, L., Bower, A.G., Collins, C.H. and Koffas, M.A. 2013. Modular optimization of multi-gene pathways for fatty acids production in E.coli. Nature Communications. 4: (1409): 1-8. DOI: 10.1038/ncomms2425<br>
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<p><b>Further analysis</b><br>
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The data obtained from the LC-MS was enormous. 2721 metabolites were detected in the samples, and so we filtered this down to the 43 fatty acids and phospholipids incorporating the compounds we were interested in (oleic acid and linoleic acid). A heat map was generated (seen above) showing the abundances of these 43 phospholipids and fatty acids in the 17 samples we run on the LC-MS. From this heatmap the diversity of detected compounds and high dynamic range can be seen. For the characterisation of our delta 12 desaturase BioBrick construct, a focus on the individual compounds of high intensity was necessary. An example of this can be seen in the bar chart above. Here you can see that, when fed with oleic acid (to a total w/v concentration of 0.1% and 0.5%), the amount of linoleic acid found within this representative phospholipid is much higher in the delta 12 desaturase expression strain than in the wild-type DH5-alpha strain. From this data we can confidently conclude that the delta 12 desaturase is converting the substrate oleic acid into the linoleic acid. Currently, the linoleic is incorporated into a phospholipid, but expression of the tesA gene (biobricked previously) would cleave the compound away to give free fatty acid. <br>
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<br>
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<b>A work flow of the methods we used to get from sample to data analysis can be seen above.</b>
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</p>
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[3]. Carson, M., Johnson, D.H., McDonald, H., Brouillette C. and Delucas, L.J. (2007) His-tag impact on structure. Acta Crystallogr D Biol Crystallogr. 63(3):295-301. <br>
 
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[4]. Chant, A., Kraemer-Pecore, C.M., Watkin, R. and Kneale G.G. 2005. Attachment of a histidine tag to the minimal zinc finger protein of the Aspergillus nidulans gene regulatory protein AreA causes a conformational change at the DNA-binding site. Protein Expr Purif. 39(2):152-9.<br>
 
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[5]. Freydank, A.C., Brandt, W. and Dräger, B. 2008. Protein structure modeling indicates hexahistidine-tag interference with enzyme activity. Proteins. 72(1):173-83. doi: 10.1002/prot.21905.<br>
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[6]. Klose, J., Wendt, N., Kubald, S., Krause, E., Fechner, K., Beyermann, M., Bienert, M., Rudolph, R. and Rothemund, S. 2004. Hexa-histidin tag position influences disulfide structure but not binding behavior of in vitro folded N-terminal domain of rat corticotropin-releasing factor receptor type 2a. Protein Sci. 13(9):2470-5. <br>
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[7]. Woestenenk, E.A., Hammarström M., Van Den Berg, S., Härd, T. and Berglund, H. 2004 His tag effect on solubility of human proteins produced in Escherichia coli: a comparison between four expression vectors. J Struct Funct Genomics. <br>5(3):217-29.
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            <img id="right" src="https://static.igem.org/mediawiki/2013/9/95/Delta12afmmanc.jpg" width="430" height="450"/>
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              <p id="footer"><b>Atomic Force Microscopy pictures. Left: WT DH5-alpha. Right: DH5-alpha transformed with delta 12 desaturase expression plasmid.</b><br>
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<br><br>
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[8]. Pronk, S., Páll, S., Schulz, R., Larsson, P., Bjelkmar, P., Apostolov, R., Shirts, M.R., Smith, J.C., Kasson, P.M., Van der Spoel, D., Hess, B., Lindahl, E., 2013. GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics: 1–10.<br>
 
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[9]. Frigo, M., Johnson, S.G., 2005. The Design and Implementation of FFTW3. Proceedings of the IEEE 93: 216–231.<br>
 
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[10]. Hornak, V., Abel, R., Okur, A., 2006. Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins: Structure, Function and Bioinformatics 65: 712–725.<br>
 
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[11]. Jorgensen, W.L., Chandrasekhar, J., Madura, J.D., Impey, R.W., Klein, M.L., 1983. Comparison of simple potential functions for simulating liquid water. The Journal of Chemical Physics 79: 926.<br>
 
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[12]. Berendsen, H., 1981. Interaction models for water in relation to protein hydration. Intermolecular Forces: 331–338<br>
 
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[13]. Kiwiel, K., Murty, K., 1996. Convergence of the steepest descent method for minimizing quasiconvex functions. Journal of Optimization Theory and Applications 89: 221–226.<br>
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<p><b>Atomic Force Microscopy</b><br>
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[14]. Darden, T., York, D., Pedersen, L., 1993. Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large systems. The Journal of Chemical Physics 98: 10089.<br>
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Whilst overexpression of the delta 12 desaturase led to a 4 fold increase in linoleic acid production incorporated in certain phospholipids, we had hypothesised that both overexpression of a non-native membrane protein and the resulting fatty acid composition changes would possibly alter <i>E. coli</i> membrane structure and potentially interfere growth and replication. To address this, we performed Atomic Force Microscopy (AFM) on transformed wild-type and <i>E. coli</i> transformed with our delta 12 desaturase expression construct. From this we found that there was no significant difference in the membranes between the wild-type and delta 12 desaturase expression constructs. From this we can conclude that overexpression of the non-native membrane bound delta 12 desaturase using our constitutive promoter system does not significantly alter the membranes. Therefore on an industrial scale the expression construct could be modified, such as by the addition of a stronger promoter sequence and RBS site or potentially using inducible expression systems, thus resulting in a greater production of linoleic acid.  
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[15]. Karttunen, M., Rottler, J., Vattulainen, I., Sagui, C., 2008. CHAPTER 2 Electrostatics in Biomolecular Simulations  : Where Are We Now and Where Are We Heading  ? Current Topics in Membranes 60: 49–89.<br>
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[16]. Hess, B., Bekker, H., Berendsen, H.J.C., Fraaije, J.G.E.M., 1997. LINCS: A linear constraint solver for molecular simulations. Journal of Computational Chemistry 18: 1463–1472.<br>
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[17]. Bussi, G., Donadio, D., Parrinello, M., 2007. Canonical sampling through velocity rescaling. The Journal of Chemical Physics 126: 014101.<br>
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[18]. Parrinello, M., 1981. Polymorphic transitions in single crystals: A new molecular dynamics method. Journal of Applied Physics 52: 7182.<br>
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[19]. Lindahl, E., Hess, B., Spoel, D. Van Der, 2001. GROMACS 3.0: a package for molecular simulation and trajectory analysis. Molecular Modeling Annual: 306–317.<br>
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[20] J. Lemkul, Department of Biochemistry, Virginia Tech. http://www.bevanlab.biochem.vt.edu/Pages/Personal/justin/gmx-tutorials/lysozyme/. [Last Accessed: 27/09/2013]<br> </p>
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Latest revision as of 15:53, 28 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!

We used an existing BioBrick component with Constitutive promoter and RBS

(BBa_K608002) to express our BioBrick parts:

Successful Expression of delta 9 and delta 12 desaturase, and FabA

In order to characterise our biobricks, we made use of the standard parts found in the registry. By inserting the created biobricks BBa_K1027001 and BBa_K1027002 in to BBa_K608002 (a biobrick consisting of a ribosomal binding site and a constitutive promoter), we were able to create a new construct that expressed the delta 9 desaturase, delta 12 desaturase and FabA proteins. The constructs are shown above.

Plates

Another piece of evidence suggesting that our constructs were successfully expressing our delta 9 desaturase and delta 12 desaturase enzymes was the size of the colonies grown on the plates. Pictures of the plates are shown below. On the far left is a control plate, with bacteria transformed with BBa_K608002 but with no gene inserted in front of the promoter. To the right of that are our delta 12 colonies. They are much smaller than the control, and even taking 20 hours to grow to that size as opposed to the usual 16 hours. We hypothesise that because delta 12 desaturase is a membrane-bound protein that the E. coli does not normally express, constitutively expressing it could inhibit the bacteria and slow growth. Compare this to FabA on the far right. Looking through the literature, we found that overexpression of FabA results in no significant difference in growth size or speed relative to the wild type strain (Luo et al, 2009). Compared to the control plate, we also found that the colonies grew normally.

Gel Digests

To confirm that our desired genes were in fact within the expression construct mentioned above, we carried out test digests of our ligated plasmids. Happily, when we ran our digests on an agarose gel, we saw all of the bands we would predict from the expected fragments. The gel pictures are on the right.
delta 9 desaturase: Cut with BamHI + EcoRV. Expected bands 1424 bp, 1262 bp, 99 bp.
delta 12 desaturase: Cut with BamHI, XbaI, PstI. Expected bands 2127 bp, 656 bp, 430 bp.
FabA Cut with EcoRV. Expected bands 1429 bp, 1115 bp. Cut with EcoRV, PstI. Expected bands 1153 bp, 1115 bp, 339 bp.


Improvement of existing BioBrick

Our biobrick BBa_K1027002 (delta 12 desaturase) is an improvement of the BBa_K925000 biobrick created by the St Andrews team in 2012. We improved this biobrick from the ground up, starting by taking the protein sequence from Synechocystis sp. PCC 6803. This protein sequence was reverse translated and then codon optimised for expression in E. coli. We believe our redesigned biobrick has two features that the original biobrick does not. Firstly, the original biobrick could not be transformed by iGEM HQ, whereas we have managed successful transformation (as shown by the plate pictures). Next, the St. Andrews biobrick was cloned from cyanobacteria, and so is not optimised for expression in E. coli. As St. Andrews then went on to express their delta 12 desaturase biobrick in E. coli it follows that a sequence optimised for expression in this chassis, as ours was, would perform better than the sequence naturally found in cyanobacteria.

Orbitrap LC-MS analysis

Delta 9 desaturase and delta 12 desaturase enzymes were chosen because their products, when expressed in their host organism (Synechocystis sp. PCC 6803), convert stearic acid into oleic acid, and oleic acid into linoleic acid respectively. Therefore, we fed batches of transformed DH5-alpha with 2 different concentrations of exogenous fatty acid (0.1% and 0.5% stearic acid fed to the delta 9 desaturase batch, and 0.1% and 0.5% oleic acid fed to the delta 12 desaturase batch), left the cultures growing overnight and then harvested the cells.

To analyse the metabolites extracted from both wild-type DH5-alpha and DH5-alpha expressing our delta 9 desaturase and delta 12 desaturase enzymes, we made use of the MIB’s in-house Orbitrap Liquid Chromatography - Mass Spectrometry (LC-MS). This technique was chosen because of its high mass accuracy and sensitivity. Upon analysing the most abundant metabolites extracted from our expression strains and comparing this data with the most abundant metabolites extracted from wild-type, it is apparent that a massive increase in linoleic acid (incorporated in phosphatidylethanolamines, PE) has occurred. This is demonstrated in the two figures directly below. The chromatograms produced for the delta 12 desaturase expression strains are also shown below. There is a clear difference between the wild-type E. coli fed with exogenous substrate compared with the E. coli strains expressing delta 12 desaturase. It is probable that the peak appearing around 7.9 min in the delta 12 desaturase strains is due to phospholipid incorporating 18:2 (9Z, 12Z) - linoleic acid.

Further analysis
The data obtained from the LC-MS was enormous. 2721 metabolites were detected in the samples, and so we filtered this down to the 43 fatty acids and phospholipids incorporating the compounds we were interested in (oleic acid and linoleic acid). A heat map was generated (seen above) showing the abundances of these 43 phospholipids and fatty acids in the 17 samples we run on the LC-MS. From this heatmap the diversity of detected compounds and high dynamic range can be seen. For the characterisation of our delta 12 desaturase BioBrick construct, a focus on the individual compounds of high intensity was necessary. An example of this can be seen in the bar chart above. Here you can see that, when fed with oleic acid (to a total w/v concentration of 0.1% and 0.5%), the amount of linoleic acid found within this representative phospholipid is much higher in the delta 12 desaturase expression strain than in the wild-type DH5-alpha strain. From this data we can confidently conclude that the delta 12 desaturase is converting the substrate oleic acid into the linoleic acid. Currently, the linoleic is incorporated into a phospholipid, but expression of the tesA gene (biobricked previously) would cleave the compound away to give free fatty acid.

A work flow of the methods we used to get from sample to data analysis can be seen above.

Atomic Force Microscopy
Whilst overexpression of the delta 12 desaturase led to a 4 fold increase in linoleic acid production incorporated in certain phospholipids, we had hypothesised that both overexpression of a non-native membrane protein and the resulting fatty acid composition changes would possibly alter E. coli membrane structure and potentially interfere growth and replication. To address this, we performed Atomic Force Microscopy (AFM) on transformed wild-type and E. coli transformed with our delta 12 desaturase expression construct. From this we found that there was no significant difference in the membranes between the wild-type and delta 12 desaturase expression constructs. From this we can conclude that overexpression of the non-native membrane bound delta 12 desaturase using our constitutive promoter system does not significantly alter the membranes. Therefore on an industrial scale the expression construct could be modified, such as by the addition of a stronger promoter sequence and RBS site or potentially using inducible expression systems, thus resulting in a greater production of linoleic acid.

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