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<div class="content" id="content-main"><div class="row"><div class="col col-lg-8 col-md-12"><div class="content-main"><h1 id="consortiaflux">ConsortiaFlux</h1>
<h1 id="metaflux">METAFLUX</h1>
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<h2 id="introduction">Introduction</h2>
<p>In nature, it is hard to find one strain of bacteria in an isolated region. Bacteria exist in communities called microbiome. In synthetic biology, although one strain of bacteria is used for experiments, targetted application area generally is a microbiome. This requires experiments to be conducted at application site or creation of pseudo microbiomes. There is a need for a tool that can predict effect of a synthetically engineered bacteria within a microbiome. We created a tool called Metaflux that can be used to compute community level flux-balance analysis. This tool allows users to visualize predicted effects of addition of a synthetically engineered bacteria into a defined microbiome. Additionally, we generated a detailed framework for the application of synthetically engineered bacteria in bioremediation efforts. To demonstrate the applicability of our framework, we focused on using genetically modified E.coli to remediate toluene in Albertan tailing ponds, to provide a concrete example of how this framework can be applied. Lastly, we conducted further analysis of how to choose the best application method, with respect to the technical, economic, and social aspects of the technology’s application.</p>
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<p>In nature, it is virtually impossible to find an isolated bacterial species on its own. Across most of the natural world, different bacterial species cluster together in ecologically crucial communities known as microbiomes. In constrast, synthetic biology laboratories often prepare isolated strains for later use in the natural environment. Experimentally, this would require either the creation or simulation of an isolated microbiome.</p>
 +
<p>Our iGEM team created the web-based tool &#39;ConsortiaFlux&#39; in order to run microbiome simulations. Our tool allows the user to visualize the effects of the addition of a synthetically engineered bacteria into a predefined microbiome. This visualization is generated through a process called community flux balance analysis (cFBA).</p>
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<p>In concert with our web-based tool, we genetically engineered E. coli bacteria for the purpose of bioremediation. Our engineered E. coli supplements the existing microbiome by efficiently degrading toluene, a highly toxic chemical within Alberta&#39;s oil sands tailing ponds.</p>
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<p>All aspects of our project are linked together by analysis of technical, economic, and social applications of our technology within a solid policy framework.</p>
 
<h2 id="what-is-fba-and-cfba-">What is FBA and cFBA?</h2>
 
<h2 id="what-is-fba-and-cfba-">What is FBA and cFBA?</h2>
<p>FBA stands for flux-balance analysis.It is a tool used to study genome-scale metabolic network reconstructions. By analyzing flow of metabolites within a given network, FBA computes predicted production rate of a metabolite or the growth rate. In synthehtic biology context, this becomes usefull in assesing outcomes of a transformation in silico.</p>
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<p>FBA (flux balance analysis) is a mathematical tool used to study genome-scale metabolic network reconstructions. By analyzing the flow of metabolites within a given network, FBA can be used to model the expected rates of production for metabolites, metabolic behaviour under optimal growth, and various other aspects of life processes. In a synthetic biology context, FBA is a useful means of assessing the outcomes of in silico transformations.</p>
<p>cFBA stands for community based flux-balance analysis. Briefly, it is the FBA in community level. cFBA enables predicting outcomes of a genetic modification in a bacterial community.</p>
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<p>cFBA (community based flux-balance analysis) consists of FBA at the community level. Highly interdependent microbial communities share essential metabolites, engage in complex metabolic exchanges, and collectively perform vital ecological functions. Using cFBA, the consequences of genetically modifying a particular species within the microbiome can be predicted.</p>
 
<h2 id="math-behind-the-code">Math Behind the Code</h2>
 
<h2 id="math-behind-the-code">Math Behind the Code</h2>
<p>FBA calcultes the flow of metabolites at steady state in which mass balance is not changing with time (i.e. (dx/dt)=0). In an FBA with m unique compounds and n reactions, a stoichiometric matrix(S) with size m*n represents all the reaction set. Vector fluxes to be computed are respresented by a vector v. An objective function (Z) is set to determine which flux to optimize in a given boundary. Hence following is computed linearly,</p>
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<p>FBA computes the flow of metabolites at steady state in which mass balance is not changing with time (i.e. (dx/dt)=0). In an FBA with <em>m</em> unique compounds and <em>n</em> reactions, a stoichiometric matrix(S) with size <em>m</em> * <em>n</em> represents all the reaction set. Vector fluxes to be computed are represented by a vector v. An objective function (Z) is set to determine which flux to optimize in a given boundary. Hence following is computed linearly,</p>
<pre><code>                                            Maximize     c^Tv
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<div>
                                            <span class="hljs-keyword">Subject </span>to  Sv<span class="hljs-number">=0</span>
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    $$ \begin{array}{l l}
                                            <span class="hljs-keyword">Boundaries </span> lowerbound&lt;v&lt;upperbound
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        \textrm{Maximize} &amp; c^Tv \\
</code></pre><p>(Insert Image: FBA_diagram.jpg)
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        \textrm{Subject to} &amp; Sv=0 \\
Taken from:
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        \textrm{boundaries} &amp; \textrm{lowerbound} &lt; v &lt; \textrm{upperbound}
Title:What is flux balance analysis?
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    \end{array} $$
 +
</div>
 +
 
 +
<!-- Maximize    c^Tv
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Subject to  Sv=0
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Boundaries  lowerbound<v<upperbound -->
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<p class="image-wrapper">
 +
</html> [[File:Toronto_2015_FBA_diagram.jpg]] <html>
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</p>
 +
 
 +
<p>Taken from:</p>
 +
<p>Jeffrey D Orth, Ines Thiele, Bernhard Ø Palsson. What is flux balance analysis?
 +
Nature Biotechnology. Nature Publishing Group. Mar 1 2010. Copyright © 2010,
 +
Rights Managed by Nature Publishing Group</p>
 +
<!-- Title:What is flux balance analysis?
 
Author:Jeffrey D Orth, Ines Thiele, Bernhard Ø Palsson
 
Author:Jeffrey D Orth, Ines Thiele, Bernhard Ø Palsson
 
Publication:Nature Biotechnology
 
Publication:Nature Biotechnology
 
Publisher:Nature Publishing Group
 
Publisher:Nature Publishing Group
 
Date:Mar 1, 2010
 
Date:Mar 1, 2010
Copyright © 2010, Rights Managed by Nature Publishing Group</p>
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Copyright © 2010, Rights Managed by Nature Publishing Group -->
<h2 id="proof-of-concept-a-genetically-engineered-solution-for-oil-sand-tailings">Proof-of-Concept: A Genetically Engineered Solution for Oil Sand Tailings</h2>
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<p>Note: See software section for more</p>
 +
<h2 id="proof-of-concept-a-genetically-engineered-solution-for-oil-sand-tailings">Proof-of-Concept A Genetically Engineered Solution for Oil Sand Tailings</h2>
 
<h3 id="background">Background</h3>
 
<h3 id="background">Background</h3>
<p>As conventional oil production declines in the coming decades, unconventional
+
<p>As conventional oil production declines in the coming decades, unconventional sources of oil such as bitumen will become increasingly important for supplying oil. Currently, Canada possesses the largest of these bitumen reserves in Alberta in the form of oil sands. Alberta’s oil sands have about 168 billion barrels, making it the third largest crude oil reserve in the world. The use and export of this oil significantly contributes to economic growth in Canada, and will continue to do so.</p>
sources of oil such as bitumen will become increasingly important for supplying
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oil. Currently, Canada possesses the largest of these bitumen reserves in
+
Alberta in the form of oil sands. Alberta’s oil sands have about 168 billion
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barrels, making it the third largest crude oil reserve in the world. The use and
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export of this oil significantly contributes to economic growth in Canada, and
+
will continue to do so.</p>
+
 
<h3 id="economic-impact-of-oil-sands-on-canada">Economic impact of oil sands on Canada</h3>
 
<h3 id="economic-impact-of-oil-sands-on-canada">Economic impact of oil sands on Canada</h3>
 
<ul>
 
<ul>
Line 74: Line 110:
 
<li>For every direct job generated in the Alberta oil sands, 1 additional job is generated by indirect association and 1.5 jobs by induced association in Canada.</li>
 
<li>For every direct job generated in the Alberta oil sands, 1 additional job is generated by indirect association and 1.5 jobs by induced association in Canada.</li>
 
</ul>
 
</ul>
<p>Despite this huge economic potential, the use of oil sands is raising concerns
+
<p>Despite this huge economic potential, the use of oil sands is raising concerns regarding environmental costs, especially those associated with the creation of oil sands tailings. Since the oil sand tailings are essentially a dumping site, it has a high concentration of toxic organic compounds. The Government of Alberta has a zero discharge policy for open-mined oil operations. This means that all oil sand process-affected water (OSPW) and tailings must be stored on site, which leads to the accumulation of these toxic compounds . According to a report released by WWF, this is a growing problem. From 2006 to 2009, weight percentage of toxic benzene, toluene, ethylbenzene, xylene (BTEX) and Polycyclic Aromatic Hydrocarbons (PAH) present in oil tailings have increased by 29.7% and 15.5% respectively.  Although, the oil sand tailing ponds is supposed to retain the toxic water, a recent Federal study suggests that the waste might be leaching into the ground water and contaminating the Athabasca River. It was estimated that as much as 6.5 litres might be leaching from a single pond per day. Current BTEX concentrations have already exceeded the guidelines provided by Canadian Council for Ministers of Environment (CCME) and thus are toxic to wildlife and humans in the long term.</p>
regarding environmental costs, especially those associated with the creation of
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oil sands tailings.   Since the oil sand tailings are essentially a dumping
+
site, it has a high concentration of toxic organic compounds. The Government of
+
Alberta has a zero discharge policy for open-mined oil operations. This means
+
that all oil sand process-affected water (OSPW) and tailings must be stored on
+
site, which leads to the accumulation of these toxic compounds . According to a
+
report released by WWF, this is a growing problem. From 2006 to 2009, weight
+
percentage of toxic benzene, toluene, ethylbenzene, xylene (BTEX) and Polycyclic
+
Aromatic Hydrocarbons (PAH) present in oil tailings have increased by 29.7% and
+
15.5% respectively.  Although, the oil sand tailing ponds is supposed to retain
+
the toxic water, a recent Federal study suggests that the waste might be
+
leaching into the ground water and contaminating the Athabasca River. It was
+
estimated that as much as 6.5 litres might be leaching from a single pond per
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day. Current BTEX concentrations have already exceeded the guidelines provided
+
by Canadian Council for Ministers of Environment (CCME) and thus are toxic to
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wildlife and humans in the long term.</p>
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<p>BTEX can cause damage to:</p>
 
<p>BTEX can cause damage to:</p>
 
<ul>
 
<ul>
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<ul>
 
<ul>
 
<li><p><em>Soil Vapor Extraction (SVE)</em></p>
 
<li><p><em>Soil Vapor Extraction (SVE)</em></p>
<p>SVE is a physical process that involves sucking up vapors of contaminants. This
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<p>SVE is a physical process that involves sucking up vapors of contaminants. This reduces the vapor pressure of the contaminant hence shifting the equilibrium towards the vapor phase and causing any contaminant in the solid or liquid phase to evaporate. Since the effectiveness of SVE depends strongly on:  </p>
reduces the vapor pressure of the contaminant hence shifting the equilibrium
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towards the vapor phase and causing any contaminant in the solid or liquid
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phase to evaporate. Since the effectiveness of SVE depends strongly on:  </p>
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<ul>
 
<ul>
 
<li>The contaminant’s physical and chemical properties</li>
 
<li>The contaminant’s physical and chemical properties</li>
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</li>
 
</li>
 
<li><p><em>Bioremediation using Microorganisms</em></p>
 
<li><p><em>Bioremediation using Microorganisms</em></p>
<p>This technique uses microbes in order to break down BTEX and PAH using enzymes
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<p>This technique uses microbes in order to break down BTEX compounds and PAHs using enzymes produced by local microbes. This technique is advantageous because it is cheap, occurs on site, and results in the production of non-toxic compounds such as carbon dioxide and acetate. However, the bioremediation process can be hindered as a result of any of the factors listed below:</p>
produces by the microbes. This technique is advantageous because it is: cheap,
+
occurs on site and produces non-toxic compounds such as carbon dioxide and
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acetate. However the Bioremediation process is slow because of:</p>
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<ul>
 
<ul>
 
<li>The high toxicity of the pollutant</li>
 
<li>The high toxicity of the pollutant</li>
Line 126: Line 140:
 
<li>Fewer electron acceptors.</li>
 
<li>Fewer electron acceptors.</li>
 
</ul>
 
</ul>
<p>This essentially limits the bioremediation process.</p>
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<p>The factors listed essentially limit the bioremediation process.</p>
 
</li>
 
</li>
 
</ul>
 
</ul>
 
<h3 id="our-project">Our Project</h3>
 
<h3 id="our-project">Our Project</h3>
<p>Our project therefore aims to maximize the efficiency of the bioremediation
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<p>Our project aims to maximize the efficiency of the bioremediation process by creating a synthetically engineered microorganism that can degrade toxic compounds found in oil sands tailing ponds. Our team focused specifically on toluene due to the prevalence of BTEX compounds (uncluding toluene) in oil sands tailing ponds, as well as fact that toluene is well-studied as a chemical.</p>
process by creating a synthetically engineered microorganism that can degrade
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these toxic compounds. Our team focused specifically on toluene since it is one
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of the major PAHs present in these oil sand tailings and is one of the most
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studied one’s.</p>
+
 
<h3 id="toluene-degradation-in-microbial-communities">Toluene degradation in microbial communities</h3>
 
<h3 id="toluene-degradation-in-microbial-communities">Toluene degradation in microbial communities</h3>
<p>Many microorganisms that survive in these oil sand tailings use toluene as a nutrient source. They have therefore evolved several pathways that can degrade toluene. These can be broadly divided into two categories:</p>
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<p>Many microorganisms that survive in Alberta&#39;s oil sand tailings use toluene as a nutrient source. As such, these microorganisms have naturally evolved several pathways that can degrade toluene. These degradation pathways can be broadly divided into two categories:</p>
 
<ul>
 
<ul>
 
<li>Aerobic pathways</li>
 
<li>Aerobic pathways</li>
 
<li>Anaerobic pathways</li>
 
<li>Anaerobic pathways</li>
 
</ul>
 
</ul>
<p>We focused specifically on the pathway used by <em>Pseudomonas putida F1</em> since it
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<p>We focused specifically on the pathway used by <em>Pseudomonas putida F1</em> because it is a well-studied, Biosafety Level 1 organism, and some studies suggest that it is one of the most efficient bacterial species with regards to toluene degradation. <em>P. putida F1</em> degrades toluene through an aerobic pathway. In its pathway the reactions involving <em>todC1C2BA</em>, <em>todD</em>, <em>todE</em> and <em>todF</em> are specific to <em>P. putida F1</em> and are the limiting steps of this pathway. In particular, the accumulation of 3-methylcatechol is known to have severe toxic effects for bacteria. Once 2-Hydroxy-2,4-pentadienoate is formed, it can be degraded by enzymes that can are present in most species, including <em>E.coli</em>. A plasmis for the first part of the pathway involving <em>todC1C2BA</em> and <em>todD</em> (pTDG602) has been created by Zylstra, from whom we obtained it. Finally, we chose to focus on the part of the pathway involving <em>todE</em> and <em>todF</em>, for which no well-characterized BioBrick exists.</p>
is a well-studied biosafety level 1 organism and some studies suggest it is one
+
of the most efficient at toluene degradation.   <em>P. putida F1</em> degrades toluene
+
through an aerobic pathway. In its pathway the reactions involving <em>todC1C2BA</em>,
+
<em>todD</em>, <em>todE</em> and <em>todF</em> are specific to <em>P. putida F1</em> and are the limiting
+
steps of this pathway. Once 2-Hydroxy-2,4-pentadienoate is formed it can be
+
degraded by enzymes that can are present in most species including <em>E.coli</em>.   A
+
bio brick, pTDG602, for the first part of the pathway involving <em>todC1C2BA</em> and
+
<em>todD</em> has already been created by Zylstra, from whom we then obtained it. This
+
only leaves <em>todE</em> and <em>todF</em> for which no such biobrick exists that has been
+
well characterized and so we chose to focus on that part of the pathway.</p>
+
 
<h2 id="experimental-design">Experimental Design</h2>
 
<h2 id="experimental-design">Experimental Design</h2>
 
<h3 id="our-objectives">Our Objectives</h3>
 
<h3 id="our-objectives">Our Objectives</h3>
Line 163: Line 163:
 
<h3 id="plasmid-design">Plasmid Design</h3>
 
<h3 id="plasmid-design">Plasmid Design</h3>
 
<p class="image-wrapper">
 
<p class="image-wrapper">
</html> [[undefined:Toronto_2015_plasmid.jpg]] <html>
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</html> [[File:Toronto_2015_lcrconstruct.png]] <html>
 
</p>
 
</p>
  
<p>The diagram above shows the plasmid that we designed. We used psB1C3, in green,
+
<p>The diagram above shows the plasmid that we designed. We used psB1C3, in green, for the backbone that we obtained from the iGEM registry. The plasmid includes both <em>todE</em> and <em>todF</em> that have been optimized for <em>E.coli</em> K-12 MG1655 strain. Each gene begins with a start codon followed by a His-Tag, and terminates with a stop codon. The purpose of the His-Tag is to confirm the synthesis of our enzymes once we transform <em>E.coli</em>. We added a Ribosomal binding site (RBS) before each gene and optimized it using the Salis RBS calculator. UNSs were also added before and after every gene to prevent the formation of a secondary structure.  </p>
for the backbone that we obtained from the igem registry. The plasmid includes
+
both <em>todE</em> and <em>todF</em> that have been optimized for <em>E.coli</em> K-12 MG1655 strain .
+
Each gene begins with a start codon followed by a His-Tag and terminates with a
+
stop codon. The purpose of the His-Tag is to confirm the synthesis of our
+
enzymes once we transform <em>E.coli</em>. We added a Ribosomal binding site (RBS)
+
before each gene and optimized it using the Salis RBS calculator. UNSs were also
+
added before and after every gene to prevent the formation of a secondary
+
structure.  </p>
+
 
<h3 id="experimental-details">Experimental details</h3>
 
<h3 id="experimental-details">Experimental details</h3>
<p>To create our plasmid we used Synbiota’s RDP protocol that allowed us to
+
<p>To create our plasmid, we used Synbiota’s Rapid Dna Prototyping (RDP) protocol in order to assemble our <em>todE</em> and <em>todF</em> constructs. We confirmed the identity of our constructs by using gel electrophoresis to compare our constructs against the <em>todE</em> and <em>todF</em> genes we purified from <em>P. putida F1</em>. Once the <em>todE</em> and <em>todF</em> genes were assembled, they were inserted into the psB1C3 backbone using the Ligase Chain Reaction (LCR) method. Following LCR, gel electrophoresis was once again used to ensure that our plasmid had been assembled correctly. Once our plasmid was assembled, we transformed <em>E.coli</em>, which we made chemically competent for transformation. Following the transformation, antibiotic selection was used to identify the transformed colonies. Further, to test for synthesis of the desired enzymes, the transformed colonies were lysed and tested via SDS-PAGE. After confirming enzyme production, the transformed <em>E.coli</em> was grown on media rich in 3-methylcatechol, and a colorimetric assay was used to confirm the formation of 2-hydroxy-6-keto-2,4-heptadienoate (substrate of <em>todE</em>) and 2-Hydroxy-2,4-pentadienoate (substrate of <em>todF</em>). As an aside, gas chromatography and mass spectrometry methods are also applicable to characterizing the activity of these enzymes, where time permits. Finally, we attempted to co-transform our bacteria with our custom-designed plasmid and pTDG602 in order to observe the complete degradation of toluene into carbon dioxide and water.</p>
assemble our <em>todE</em> and <em>todF</em> constructs. We confirmed the identity of our
+
</div></div><div id="tableofcontents" class="tableofcontents affix sidebar col-lg-4 hidden-xs hidden-sm hidden-md visible-lg-3"><ul class="nav">
constructs by using gel electrophoresis to compare our constructs against the
+
<li><a href="#introduction">Introduction</a></li>
<em>todE</em> and <em>todF</em> genes we purified from <em>P. putida F1</em>. Once the <em>todE</em> and
+
<li><a href="#what-is-fba-and-cfba-">What is FBA and cFBA?</a></li>
<em>todF</em> genes have been assembled they will be inserted into the psB1C3 backbone
+
<li><a href="#math-behind-the-code">Math Behind the Code</a></li>
using Ligase Chain Reaction (LCR). Gel electrophoresis will once again be used
+
<li><a href="#proof-of-concept-a-genetically-engineered-solution-for-oil-sand-tailings">Proof-of-Concept A Genetically Engineered Solution for Oil Sand Tailings</a><ul>
to ensure that our plasmid has been correctly assembled. Once our plasmid is
+
<li><a href="#background">Background</a></li>
assembled we will then transform <em>E.coli</em> that we will make chemically competent
+
<li><a href="#economic-impact-of-oil-sands-on-canada">Economic impact of oil sands on Canada</a></li>
for transformation. Antibiotic selection will be used to identify the
+
<li><a href="#current-solutions">Current Solutions</a></li>
transformed colonies. To test for the synthesis of the enzymes, the transformed
+
<li><a href="#our-project">Our Project</a></li>
colonies will be lysed and SDS-PAGE and western blotting will be used.   After
+
<li><a href="#toluene-degradation-in-microbial-communities">Toluene degradation in microbial communities</a></li>
confirming the production of the enzymes, the transformed <em>E.coli</em> will be grown
+
</ul>
on a 3-methyl catechol rich media and then a colorimetric assay will be used to
+
</li>
identify the formation of 2-Hydroxy-6-keto-2,4-heptadienoate (substrate of
+
<li><a href="#experimental-design">Experimental Design</a><ul>
<em>todE</em>); and 2-Hydroxy-2,4-pentadienoate substrate of <em>todF</em>). Also, if time
+
<li><a href="#our-objectives">Our Objectives</a></li>
permits Gas Chromatography and Mass spectrometry will also be used to
+
<li><a href="#plasmid-design">Plasmid Design</a></li>
characterize the activity of these enzymes.   Furthermore, we will co-transform
+
<li><a href="#experimental-details">Experimental details</a></li>
the bacteria with our designed plasmid and pTDG602 to observe a complete
+
</ul>
degradation of toluene to carbon dioxide and water.</p>
+
</li>
<h3 id="progress">Progress</h3>
+
</ul>
<p>We have currently assembled our <em>todE</em> and <em>todF</em> genes using the symbiota
+
</div></div></div>
protocol. To add the <em>todE</em> and <em>todF</em> genes into the plasmid we tried to use
+
LCR; however, due to contamination it did not work hence we used Gibson to
+
assemble it. Currently, we are in the process of transforming our <em>E.coli</em> with
+
our assembled plasmid after which we will proceed to verify the production of
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our enzymes and to characterize their activity.  </p>
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Latest revision as of 04:31, 20 November 2015

ConsortiaFlux

Introduction

In nature, it is virtually impossible to find an isolated bacterial species on its own. Across most of the natural world, different bacterial species cluster together in ecologically crucial communities known as microbiomes. In constrast, synthetic biology laboratories often prepare isolated strains for later use in the natural environment. Experimentally, this would require either the creation or simulation of an isolated microbiome.

Our iGEM team created the web-based tool 'ConsortiaFlux' in order to run microbiome simulations. Our tool allows the user to visualize the effects of the addition of a synthetically engineered bacteria into a predefined microbiome. This visualization is generated through a process called community flux balance analysis (cFBA).

In concert with our web-based tool, we genetically engineered E. coli bacteria for the purpose of bioremediation. Our engineered E. coli supplements the existing microbiome by efficiently degrading toluene, a highly toxic chemical within Alberta's oil sands tailing ponds.

All aspects of our project are linked together by analysis of technical, economic, and social applications of our technology within a solid policy framework.

What is FBA and cFBA?

FBA (flux balance analysis) is a mathematical tool used to study genome-scale metabolic network reconstructions. By analyzing the flow of metabolites within a given network, FBA can be used to model the expected rates of production for metabolites, metabolic behaviour under optimal growth, and various other aspects of life processes. In a synthetic biology context, FBA is a useful means of assessing the outcomes of in silico transformations.

cFBA (community based flux-balance analysis) consists of FBA at the community level. Highly interdependent microbial communities share essential metabolites, engage in complex metabolic exchanges, and collectively perform vital ecological functions. Using cFBA, the consequences of genetically modifying a particular species within the microbiome can be predicted.

Math Behind the Code

FBA computes the flow of metabolites at steady state in which mass balance is not changing with time (i.e. (dx/dt)=0). In an FBA with m unique compounds and n reactions, a stoichiometric matrix(S) with size m * n represents all the reaction set. Vector fluxes to be computed are represented by a vector v. An objective function (Z) is set to determine which flux to optimize in a given boundary. Hence following is computed linearly,

$$ \begin{array}{l l} \textrm{Maximize} & c^Tv \\ \textrm{Subject to} & Sv=0 \\ \textrm{boundaries} & \textrm{lowerbound} < v < \textrm{upperbound} \end{array} $$

File:Toronto 2015 FBA diagram.jpg

Taken from:

Jeffrey D Orth, Ines Thiele, Bernhard Ø Palsson. What is flux balance analysis? Nature Biotechnology. Nature Publishing Group. Mar 1 2010. Copyright © 2010, Rights Managed by Nature Publishing Group

Note: See software section for more

Proof-of-Concept A Genetically Engineered Solution for Oil Sand Tailings

Background

As conventional oil production declines in the coming decades, unconventional sources of oil such as bitumen will become increasingly important for supplying oil. Currently, Canada possesses the largest of these bitumen reserves in Alberta in the form of oil sands. Alberta’s oil sands have about 168 billion barrels, making it the third largest crude oil reserve in the world. The use and export of this oil significantly contributes to economic growth in Canada, and will continue to do so.

Economic impact of oil sands on Canada

  • Total GDP impacts of all oil sands investment, re-investment and operating revenues is estimated to be $3,865 billion for Canada.
  • Oil sands related direct employment in Alberta is expected to continue growing from the current level (2014) of 146,000 jobs to a peak of 256,000 jobs in 2024.
  • For every direct job generated in the Alberta oil sands, 1 additional job is generated by indirect association and 1.5 jobs by induced association in Canada.

Despite this huge economic potential, the use of oil sands is raising concerns regarding environmental costs, especially those associated with the creation of oil sands tailings. Since the oil sand tailings are essentially a dumping site, it has a high concentration of toxic organic compounds. The Government of Alberta has a zero discharge policy for open-mined oil operations. This means that all oil sand process-affected water (OSPW) and tailings must be stored on site, which leads to the accumulation of these toxic compounds . According to a report released by WWF, this is a growing problem. From 2006 to 2009, weight percentage of toxic benzene, toluene, ethylbenzene, xylene (BTEX) and Polycyclic Aromatic Hydrocarbons (PAH) present in oil tailings have increased by 29.7% and 15.5% respectively. Although, the oil sand tailing ponds is supposed to retain the toxic water, a recent Federal study suggests that the waste might be leaching into the ground water and contaminating the Athabasca River. It was estimated that as much as 6.5 litres might be leaching from a single pond per day. Current BTEX concentrations have already exceeded the guidelines provided by Canadian Council for Ministers of Environment (CCME) and thus are toxic to wildlife and humans in the long term.

BTEX can cause damage to:

  • Liver
  • Kidneys
  • Eyes
  • Central Nervous System

Current Solutions

The current solutions for BTEX and PAH contamination includes:

  • Soil Vapor Extraction (SVE)

    SVE is a physical process that involves sucking up vapors of contaminants. This reduces the vapor pressure of the contaminant hence shifting the equilibrium towards the vapor phase and causing any contaminant in the solid or liquid phase to evaporate. Since the effectiveness of SVE depends strongly on:

    • The contaminant’s physical and chemical properties
    • Temperature in the subsurface
    • Soil properties

    So SVE is not suitable for all compounds and under all conditions.

  • Bioremediation using Microorganisms

    This technique uses microbes in order to break down BTEX compounds and PAHs using enzymes produced by local microbes. This technique is advantageous because it is cheap, occurs on site, and results in the production of non-toxic compounds such as carbon dioxide and acetate. However, the bioremediation process can be hindered as a result of any of the factors listed below:

    • The high toxicity of the pollutant
    • The high concentration of the pollutant
    • Low solubility of the pollutant
    • Low availability of nutrients in the soil
    • Low concentration of dissolved Oxygen
    • Fewer electron acceptors.

    The factors listed essentially limit the bioremediation process.

Our Project

Our project aims to maximize the efficiency of the bioremediation process by creating a synthetically engineered microorganism that can degrade toxic compounds found in oil sands tailing ponds. Our team focused specifically on toluene due to the prevalence of BTEX compounds (uncluding toluene) in oil sands tailing ponds, as well as fact that toluene is well-studied as a chemical.

Toluene degradation in microbial communities

Many microorganisms that survive in Alberta's oil sand tailings use toluene as a nutrient source. As such, these microorganisms have naturally evolved several pathways that can degrade toluene. These degradation pathways can be broadly divided into two categories:

  • Aerobic pathways
  • Anaerobic pathways

We focused specifically on the pathway used by Pseudomonas putida F1 because it is a well-studied, Biosafety Level 1 organism, and some studies suggest that it is one of the most efficient bacterial species with regards to toluene degradation. P. putida F1 degrades toluene through an aerobic pathway. In its pathway the reactions involving todC1C2BA, todD, todE and todF are specific to P. putida F1 and are the limiting steps of this pathway. In particular, the accumulation of 3-methylcatechol is known to have severe toxic effects for bacteria. Once 2-Hydroxy-2,4-pentadienoate is formed, it can be degraded by enzymes that can are present in most species, including E.coli. A plasmis for the first part of the pathway involving todC1C2BA and todD (pTDG602) has been created by Zylstra, from whom we obtained it. Finally, we chose to focus on the part of the pathway involving todE and todF, for which no well-characterized BioBrick exists.

Experimental Design

Our Objectives

  • To design and construct a plasmid that contains both todE and todF.
  • To transform E.coli with our created plasmid.
  • To confirm the synthesis of todE and todF.
  • To characterize the activity of todE and todF (if time permits).
  • To co-transform E.coli with both pTDG602 and our assembled plasmid (if time permits).

Plasmid Design

Toronto 2015 lcrconstruct.png

The diagram above shows the plasmid that we designed. We used psB1C3, in green, for the backbone that we obtained from the iGEM registry. The plasmid includes both todE and todF that have been optimized for E.coli K-12 MG1655 strain. Each gene begins with a start codon followed by a His-Tag, and terminates with a stop codon. The purpose of the His-Tag is to confirm the synthesis of our enzymes once we transform E.coli. We added a Ribosomal binding site (RBS) before each gene and optimized it using the Salis RBS calculator. UNSs were also added before and after every gene to prevent the formation of a secondary structure.

Experimental details

To create our plasmid, we used Synbiota’s Rapid Dna Prototyping (RDP) protocol in order to assemble our todE and todF constructs. We confirmed the identity of our constructs by using gel electrophoresis to compare our constructs against the todE and todF genes we purified from P. putida F1. Once the todE and todF genes were assembled, they were inserted into the psB1C3 backbone using the Ligase Chain Reaction (LCR) method. Following LCR, gel electrophoresis was once again used to ensure that our plasmid had been assembled correctly. Once our plasmid was assembled, we transformed E.coli, which we made chemically competent for transformation. Following the transformation, antibiotic selection was used to identify the transformed colonies. Further, to test for synthesis of the desired enzymes, the transformed colonies were lysed and tested via SDS-PAGE. After confirming enzyme production, the transformed E.coli was grown on media rich in 3-methylcatechol, and a colorimetric assay was used to confirm the formation of 2-hydroxy-6-keto-2,4-heptadienoate (substrate of todE) and 2-Hydroxy-2,4-pentadienoate (substrate of todF). As an aside, gas chromatography and mass spectrometry methods are also applicable to characterizing the activity of these enzymes, where time permits. Finally, we attempted to co-transform our bacteria with our custom-designed plasmid and pTDG602 in order to observe the complete degradation of toluene into carbon dioxide and water.