Team:UCL/Project/Xenobiology

From 2014.igem.org

(Difference between revisions)
 
(72 intermediate revisions not shown)
Line 5: Line 5:
<div id="BPimagewrapperHeader">
<div id="BPimagewrapperHeader">
<img src="https://static.igem.org/mediawiki/2014/a/a2/UCL2014_Xenobanner_rising-11.jpg" width="100%" height="100%" alt="Xenobiology" /></div>
<img src="https://static.igem.org/mediawiki/2014/a/a2/UCL2014_Xenobanner_rising-11.jpg" width="100%" height="100%" alt="Xenobiology" /></div>
-
 
-
<div style="padding-left:8%;padding-right:8%;">
 
-
<h2>The ultimate biosafety tool</h2>
 
-
</div>
 
<div class="textArena">
<div class="textArena">
<!--- This is the coding for the tabs (ask sanjay before altering this) --->
<!--- This is the coding for the tabs (ask sanjay before altering this) --->
 +
<ul class="tabs">
<ul class="tabs">
-
     <li><a href="#view1">Xeno</a></li>
+
     <li><a href="#view1">Introduction</a></li>
-
     <li><a href="#view2">Azoreductase</a></li>
+
     <li><a href="#view2">Strategies</a></li>
-
     <li><a href="#view3">Laccase</a></li>
+
     <li><a href="#view3">Cofactors</a></li>
-
     <li><a href="#view4">Lignin Peroxidase</a></li>
+
     <li><a href="#view4">XenoRank</a></li>
-
     <li><a href="#view5">Bacterial Peroxidases</a></li>
+
     <li><a href="#view5">Xeno-quinones</a></li>
-
     <li><a href="#view6">ispB asRNA</a></li>
+
     <li><a href="#view6">Silencing</a></li>
-
     <li><a href="#view7">Nuclease</a></li>
+
     <li><a href="#view7">References</a></li>
</ul>
</ul>
<div class="tabcontents">
<div class="tabcontents">
<!--- This is the overview section --->
<!--- This is the overview section --->
-
<div id="view1"><div class="textTitle"><h4>Our BioBricks & how they lead to azo degradation</h4></div><br>
+
<div id="view1"><div class="textTitle"><h4>Xenobiology - The Ultimate Biosafety Tool</h4></div><br>
-
<a data-tip="true" class="top large" data-tip-content="Here's Tanel doing some pipetting in our lab!" href="javascript:void(0)" style="width: 25%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/c/c9/UCLTANELPIPETTING.JPG" style="max-width: 100%;"></a>
+
-
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately-->
+
-
<p>We plan to create a complete synthetic azo dye decolourising device in <em>E. coli</em> which incorporates several different independent enzymes that act on azo dyes and their breakdown products. After evaluating their individual breakdown characteristics, we aim to investigate the potential synergistic action of these enzymes in a single synthetic <em>E. coli</em> device and design a <a data-tip="true" class="top large" data-tip-content="We developed a novel platform for industrial scale sustainable bioremediation." href="https://2014.igem.org/Team:UCL/Science/Bioprocessing"><b>bioprocess</b></a> which could be used to upscale the method to an industrial context. </p>
+
-
<a data-tip="true" class="top large" data-tip-content="Can you guess which one is the RFP BioBrick?" href="javascript:void(0)" style="width: 20%;float: left;margin-top:2%; margin-right:2%"><img src="https://static.igem.org/mediawiki/2014/c/c0/UCLTANELHOLDINGBIOBRICK.jpg" style="max-width: 100%;"></a>
+
-
<br>
+
-
In an industrial setting, these enzymes would work sequentially in a bioreactor with preset dynamic conditions. First, azoreductase will <a data-tip="true" class="top large" data-tip-content="Via a double reduction using NADPH as a cofactor." href="javascript:void(0)"><b>cleave the azo-bond (N=N)</b></a>, producing a series of highly toxic aromatic amines. Then, these compounds will be oxidised by lignin peroxidase, laccase and bacterial peroxidases, completing decolourisation and decreasing <a data-tip="true" class="top large" data-tip-content="To the point that the final products of the process are less toxic than the intact dyes themselves." href="javascript:void(0)"><b>toxicity levels</b></a>.
+
-
<br><br>
+
-
The complementary action of azoreductase, lignin peroxidase, laccase, and bacterial peroxidases will be studied in order to find out the best possible approach of sequential reaction, and this core degradation module will be extrapolated to other areas such as BioArt projects and work on <a data-tip="true" class="top large" data-tip-content="Trying to set up the foundations for a synthetic ecology." href="javascript:void(0)"><b>algal-bacterial symbiosis</b></a>.<br><br><br></p>
+
-
</div>
+
-
 
+
-
<!--- This is the first biobrick --->
+
-
<div id="view2"><div class="textTitle"><h4>Azoreductase (BBa_K1336000)</h4></div><br>
+
-
<!-- This is the biobrick image -->
+
-
<a data-tip="true" class="top large" data-tip-content="This diagram explains the basic construct of the BioBrick, the only part that changes is the selected function itself; in this case attributed to BBa_K1336000." href="javascript:void(0)" style="width: 30%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/d/dd/BBa_K1336000.png" style="max-width: 100%;"></a>
+
-
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately-->
+
-
<p>This non-specific enzyme was isolated from <em>Bacillus subtilis</em>, although it is also found in <a data-tip="true" class="top large" data-tip-content="Including those inhabiting the human intestine!" href="javascript:void(0)"><b>other bacterial species</b></a>. It starts the degradation of azo dyes by cleaving the <a data-tip="true" class="top large" data-tip-content="A bond composed of two nitrogens linked by a double bond (N=N), characteristic of all azo dyes." href="javascript:void(0)"><b>azo bond</b></a>. <br><br>The products of this cleavage varies greatly among different dyes, but are generally aromatic amines. This azo cleavage does not only occur with azo dyes, but also with other molecules like <a data-tip="true" class="top large" data-tip-content="A drug that is broken down in the gut to release compounds that fight bowel disease and arthritis." href="javascript:void(0)"><b>Sulfasalazine</b></a>. We will isolate this enzyme from <em>B. subtilis</em> and convert it to BioBrick format via polymerase chain reaction (PCR).</p><br><br>
+
-
<div class="textTitle"><h4>Azoreductase 1B6 (BBa_K1336001)</h4></div><br>
+
-
<!-- This is the biobrick image -->
+
-
<a data-tip="true" class="top large" data-tip-content="This diagram explains the basic construct of the BioBrick, the only part that changes is the selected function itself; in this case attributed to BBa_K1336001." href="javascript:void(0)" style="width: 30%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/5/5d/UCLBBAzo1b6.png" style="max-width: 100%;"></a>
+
-
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately-->
+
-
<p>Another azoreductase that we will be using is isolated from <em>Pseudomonas aeruginosa</em>. It functions in the same way as Azoreductase R1 -  cleaving the azo bond - but it is intended to work complementary with it, in order to cover a wider spectrum of dyes more efficiently. <br><br>Like the previous azoreductase, this BioBrick will be constructed using PCR. A promoter and a ribosomal binding site (RBS) will then be added to create a functioning composite device. </p><br>
+
-
</div>
+
-
 
+
-
<!--- This is the second biobrick --->
+
-
<div id="view3"><div class="textTitle"><h4>Spore Coat Protein Laccase (BBa_K1336002)</h4></div><br>
+
-
<!-- This is the biobrick image -->
+
-
<a data-tip="true" class="top large" data-tip-content="This diagram explains the basic construct of the BioBrick, the only part that changes is the selected function itself; in this case attributed to BBa_K1336001." href="javascript:void(0)" style="width: 30%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/5/5d/UCLBBAzo1b6.png" style="max-width: 100%;"></a>
+
-
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately-->
+
-
<p>Another azoreductase that we will be using is isolated from <em>Pseudomonas aeruginosa</em>. It functions in the same way as Azoreductase R1 -  cleaving the azo bond - but it is intended to work complementary with it, in order to cover a wider spectrum of dyes more efficiently. <br><br>Like the previous azoreductase, this BioBrick will be constructed using PCR. A promoter and a ribosomal binding site (RBS) will then be added to create a functioning composite device. </p><br>
+
-
<div class="textTitle"><h4>Laccase (BBa_K729006)</h4></div><br>
+
-
<!-- This is the biobrick image -->
+
-
<a data-tip="true" class="top large" data-tip-content="This diagram explains the basic construct of the BioBrick, the only part that changes is the selected function itself; in this case attributed to BBa_K1336001." href="javascript:void(0)" style="width: 30%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/5/5d/UCLBBAzo1b6.png" style="max-width: 100%;"></a>
+
-
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately-->
+
-
<p>Another azoreductase that we will be using is isolated from <em>Pseudomonas aeruginosa</em>. It functions in the same way as Azoreductase R1 -  cleaving the azo bond - but it is intended to work complementary with it, in order to cover a wider spectrum of dyes more efficiently. <br><br>Like the previous azoreductase, this BioBrick will be constructed using PCR. A promoter and a ribosomal binding site (RBS) will then be added to create a functioning composite device. </p><br>
+
-
</div>
+
-
 
+
-
<!--- This is the third biobrick --->
+
-
<div id="view4"><div class="textTitle"><h4>Lignin Peroxidase (BBa_K500000)</h4></div><br>
+
-
<a data-tip="true" class="top large" data-tip-content="This diagram explains the basic construct of the BioBrick, the only part that changes is the selected function itself; in this case attributed to BBa_K1336003." href="javascript:void(0)" style="width: 30%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/7/78/UCLBBLigningperoxidase.png" style="max-width: 100%;"></a>
+
-
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately-->
+
-
<p>Usually found in white-rot fungi species, its main function in nature is to participate in lignin-degrading processes by these organisms. However, it has also been found to play a role in <a data-tip="true" class="top large" data-tip-content="Using oxidative processes." href="javascript:void(0)"><b>azo dye degradation and decolourisation</b></a>. <br><br>This enzyme, like laccase, would be incorporated in the second step of the reaction to oxidise the products of the azo bond cleavage, in order to achieve greater detoxification. The sequence for the enzyme will be ordered and synthesised, including the BioBrick prefix and suffix. Again, it will function together with a promoter and a RBS.</p><br>
+
-
</div>
+
-
 
+
-
<!--- This is the fourth biobrick --->
+
-
<div id="view5"><div class="textTitle"><h4><em>Bacillus subtilis</em> dye-decolorizing peroxidase (BsDyP) (BBa_K1336003)</h4></div><br>
+
-
<a data-tip="true" class="top large" data-tip-content="This diagram explains the basic construct of the BioBrick, the only part that changes is the selected function itself; in this case attributed to BBa_K1336004." href="javascript:void(0)" style="width: 30%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/a/ad/UCLBBBsdyp.png" style="max-width: 100%;"></a>
+
-
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately-->
+
-
<p>Found in <em>B. subtilis</em>, the physiological function of this newly discovered enzyme is still unclear, although it has shown effectiveness in degrading lignin and azo dyes, which makes it useful for us. It is not as effective as PpDyP for most compounds, but very efficient in degrading ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)). <br><br>The BioBrick will be constructed via PCR.</p><br><br>
+
-
<div class="textTitle"><h4><em>Pseudomonas putida</em> MET94 dye-decolorizing peroxidase (PpDyP) (BBa_K1336004)</h4></div><br>
+
-
<a data-tip="true" class="top large" data-tip-content="This diagram explains the basic construct of the BioBrick, the only part that changes is the selected function itself; in this case attributed to BBa_K1336005." href="javascript:void(0)" style="width: 30%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/9/9c/UCLBBPpdyp.png" style="max-width: 100%;"></a>
+
-
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately-->
+
-
<p>This enzyme is found in <em>P. putida</em>. Although it is relatively novel, and has not yet been studied in detail, it seem to be an extremely versatile and powerful biocatalyst; it oxidises a wide <a data-tip="true" class="top large" data-tip-content="Such as azo dyes, anthraquinones, phenolic compounds, manganese or veratryl alcohol." href="javascript:void(0)"><b>variety of substrates</b></a> very efficiently. This will broaden the <a data-tip="true" class="top large" data-tip-content="Going further just azo dyes!" href="javascript:void(0)"><b>spectrum of action</b></a> of our decolourising device, and thus being able to degrade other toxic compounds typically found in industrial wastewaters. <br><br>This BioBrick will be constructed via PCR.</p><br>
+
-
</div>
+
-
 
+
-
<!--- This is the fifth biobrick --->
+
-
<div id="view6"><div class="textTitle"><h4>Octaprenyl Diphosphate Synthase (ispB) (BBa_K1336005)</h4></div><br>
+
-
<a data-tip="true" class="top large" data-tip-content="This diagram explains the basic construct of the BioBrick, the only part that changes is the selected function itself; in this case attributed to BBa_K1336005." href="javascript:void(0)" style="width: 30%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/9/9c/UCLBBPpdyp.png" style="max-width: 100%;"></a>
+
-
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately-->
+
-
<p>This enzyme is found in <em>P. putida</em>. Although it is relatively novel, and has not yet been studied in detail, it seem to be an extremely versatile and powerful biocatalyst; it oxidises a wide <a data-tip="true" class="top large" data-tip-content="Such as azo dyes, anthraquinones, phenolic compounds, manganese or veratryl alcohol." href="javascript:void(0)"><b>variety of substrates</b></a> very efficiently. This will broaden the <a data-tip="true" class="top large" data-tip-content="Going further just azo dyes!" href="javascript:void(0)"><b>spectrum of action</b></a> of our decolourising device, and thus being able to degrade other toxic compounds typically found in industrial wastewaters. <br><br>This BioBrick will be constructed via PCR.</p><br>
+
-
</div>
+
-
 
+
-
<!--- This is the fifth biobrick --->
+
-
<div id="view7"><div class="textTitle"><h4>Extracellular Nuclease (nucB) (BBa_K729004)</h4></div><br>
+
-
<a data-tip="true" class="top large" data-tip-content="This diagram explains the basic construct of the BioBrick, the only part that changes is the selected function itself; in this case attributed to BBa_K1336005." href="javascript:void(0)" style="width: 30%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/9/9c/UCLBBPpdyp.png" style="max-width: 100%;"></a>
+
-
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately-->
+
-
<p>This enzyme is found in <em>P. putida</em>. Although it is relatively novel, and has not yet been studied in detail, it seem to be an extremely versatile and powerful biocatalyst; it oxidises a wide <a data-tip="true" class="top large" data-tip-content="Such as azo dyes, anthraquinones, phenolic compounds, manganese or veratryl alcohol." href="javascript:void(0)"><b>variety of substrates</b></a> very efficiently. This will broaden the <a data-tip="true" class="top large" data-tip-content="Going further just azo dyes!" href="javascript:void(0)"><b>spectrum of action</b></a> of our decolourising device, and thus being able to degrade other toxic compounds typically found in industrial wastewaters. <br><br>This BioBrick will be constructed via PCR.</p><br>
+
-
</div>
+
-
 
+
-
 
+
-
</div>
+
-
   
+
-
<p>
+
-
<i>“Any technological advance can be dangerous. Fire was dangerous from the start, and so (even more so) was speech - and both are still dangerous to this day - but human beings would not be human without them.” - Isaac Asimov</i></p><br/>
+
-
 
+
-
<p>
+
-
The wide use of genetically modified organisms causes concerns on how they will interact in the natural environment. In particular could the genetically modified microbes escape our constrains, and outcompete the organisms found in the natural ecosystem? Could the DNA we inserted into a specific bacteria be transmitted, with unknown spread of information?
+
<p>The wide use of genetically modified organisms causes concerns on how they will interact in the natural environment. In particular could the genetically modified microbes escape our constrains, and outcompete the organisms found in the natural ecosystem? Could the DNA we inserted into a specific bacteria be transmitted, with unknown spread of information?
<br>
<br>
Since the early days of genetic engineering we had to reflect on biosafety strategies to control these risks, and synthetic biology is bringing these concerns to another level: the more we tinker with biology, the more our biosafety needs to be bullet-proof.
Since the early days of genetic engineering we had to reflect on biosafety strategies to control these risks, and synthetic biology is bringing these concerns to another level: the more we tinker with biology, the more our biosafety needs to be bullet-proof.
<br/><br/>
<br/><br/>
Xenobiology implements the term "synthetic" by creating organisms that are unable to survive in the natural environment and necessitate an artificial intervention from man to exist. It aims to create a synthetic "man-made" version of Biology, that respects the definition of life, but is based on entirely different mechanisms to function. The biochemistry of a xeno-organism uses new XNAs, genetic codes and cofactors different from the ones explored by Biology and is therefore incompatible with other forms of life. This allows a much higher level of control: a xeno-organism will not be able to find the xenocompounds in the natural environmentnor will be able to use bacterial communication systems.
Xenobiology implements the term "synthetic" by creating organisms that are unable to survive in the natural environment and necessitate an artificial intervention from man to exist. It aims to create a synthetic "man-made" version of Biology, that respects the definition of life, but is based on entirely different mechanisms to function. The biochemistry of a xeno-organism uses new XNAs, genetic codes and cofactors different from the ones explored by Biology and is therefore incompatible with other forms of life. This allows a much higher level of control: a xeno-organism will not be able to find the xenocompounds in the natural environmentnor will be able to use bacterial communication systems.
 +
</p>
<br/><br/>
<br/><br/>
-
We explored this possibility with the longer term vision of creating an <i>X. coli</i> which lives is metabolically dependent on azo dyes. An alien form of life, different from the one we know, will merge synthetic chemistry with synthetic biology - allowing the remediate the damage that the first one caused and making the remediating agent dependent on the toxic compounds. This system would be completely incompatible and invisible to regular biology, now we can ask: is non-biological life safe enough?
+
<div class="SCJBBHIGHLIGHT">
-
</p>
+
<p>We explored this possibility with the longer term vision of creating an <i>X. coli</i> which is metabolically dependent on azo dyes. An alien form of life, different from the one we know, will merge synthetic chemistry with synthetic biology - allowing the remediate the damage that the first one caused and making the remediating agent dependent on the toxic compounds. This system would be completely incompatible and invisible to regular biology, now we can ask: is non-biological life safe enough?
-
<br><br>
+
</p></div>
-
</div>
 
-
<!--<h3> Biological vs. Xenobiological strategies </h3>
 
-
Biological strategies explore biology implementing use suicide mechanisms, restriction enzymes and auxotrophies to control bacteria and bacterial communication. Our biosafety strategy is exploring the regions outside of Biology, with the ultimate goal of bringing Biology to a parallel domain where it does not interact with our own one. Three different firewalls can be used:
 
 +
<br/>
-
Why tinkering with our same Biology when we can create a new on, at the same time biology and technology, that we can control at a much higher level?-->
+
</div>
 +
 
 +
<!--- This is the first biobrick --->
 +
<div id="view2"><div class="textTitle"><h4>Biological vs. Xenobiological Strategies</h4></div><br>
-
<div class="biovsxenoBig">
+
<p>Biosafety strategies have so far explored biology to implement clever control mechanisms to control. They investigated various strategies that allow to kill bacteria when needed or that hinder genetic information to spread among different organisms.
-
    <div class="biovsxenoSmall1">
+
-
        <h3 class="shortMargin"> Biological vs. Xenobiological strategies </h3>
+
-
            <p class="edText1">
+
-
Biosafety strategies have so far explored biology to implement clever control mechanisms to control. They investigated various strategies that allow to kill bacteria when needed or that hinder genetic information to spread among different organisms.
+
Our biosafety strategy is exploring the regions outside of Biology, with the ultimate goal of bringing Biology to a parallel domain where it does not interact with our own one. Why tinkering with our same Biology when we can create a new on, at the same time biology and technology, that we can control at a much higher level?
Our biosafety strategy is exploring the regions outside of Biology, with the ultimate goal of bringing Biology to a parallel domain where it does not interact with our own one. Why tinkering with our same Biology when we can create a new on, at the same time biology and technology, that we can control at a much higher level?
-
        </p>
+
</p>
-
    </div>
+
<h5>Biological Strategies</h5>
-
    <div class="bioSmall2">
+
 
-
        <div id="bxleftHand2">           
+
<p>The biosafety mechanism is added to the system as additional layers of protection, the most explored are:
-
            <p class="shortMargin"> <h3> <center>Biological strategies </center></h3>
+
<ol>
-
            The biosafety mechanism is added to the system as additional layers of protection, the most explored are:
+
  <li>Restriction enzyme systems: autodestruction of the transformed plasmid when task ended </li>
-
                <ol>
+
  <li>Semantic containment: different meaning of stop codon, other bacteria will read as stop e.g. amber codon </li>
-
                <li>Restriction enzyme systems: autodestruction of the transformed plasmid when task ended </li>
+
  <li>Auxotrophy: knock-out of biosynthesis of a key naturally produced compound that needs to be provided in the media </li>
-
                <li>Semantic containment: different meaning of stop codon, other bacteria will read as stop e.g. amber codon </li>
+
  <li>Suicide system: bacteria die when finished its task/changes environment e.g. toxin/antitoxin where the bacteria stops producing antitoxin when triggered hence dies</li>
-
                <li>Auxotrophy: knock-out of biosynthesis of a key naturally produced compound that needs to be provided in the media </li>
+
</ol>
-
                <li>Suicide system: bacteria die when finished its task/changes environment e.g. toxin/antitoxin where the bacteria stops producing antitoxin when triggered hence dies</li>
+
-
                </ol>
+
-
            </div>
+
-
        <div id="bxrightHand2">
+
-
        <p class="shortMargin"> <h3> <center> Xenobiological strategies</center> </h3>
+
-
                The safety mechanism embedded is into the system on three different levels:
+
-
                <ol>
+
-
                <li>Genetic Firewall: Use of XNAs, incompatible with other organisms and synthetic nucleotides not found in nature</li>
+
-
                <li>Semantic Firewall: Genetic code has a different meaning than the natural code, all the codons code for a different amino acid from the standard table and could code for non-natural amino acids as well</li>
+
-
                <li>Metabolic Firewall: A Synthetic auxotrophy that uses a xenobiotic compound as key cofactor/amino acid which the bacteria is unable to produce or find in the natural enviroment </li>
+
-
                </ol>
+
-
            </div>
+
-
    </div>
+
-
</div>
+
-
    <div class="designXeno">
+
<h5>Xenobiological Strategies</h5>
-
        <h3 class="shortMargin"> Designing Xeno-Coli</h3>
+
<p>The safety mechanism embedded is into the system on three different levels:
-
            <p class="edText1">
+
<ol>
-
We aim to engineer the bacteria to utilise the synthetic dyes - a completely xenobiotic compound - as the key cofactor in respiration, substituting quinones in the electron transport chain. <br>  
+
  <li>Genetic Firewall: Use of XNAs, incompatible with other organisms and synthetic nucleotides not found in nature</li>
 +
  <li>Semantic Firewall: Genetic code has a different meaning than the natural code, all the codons code for a different amino acid from the standard table and could code for non-natural amino acids as well</li>
 +
  <li>Metabolic Firewall: A Synthetic auxotrophy that uses a xenobiotic compound as key cofactor/amino acid which the bacteria is unable to produce or find in the natural enviroment </li>
 +
</ol>
 +
 
 +
<h5>Designing Xeno-Coli</h5>
 +
<p>We aim to engineer the bacteria to utilise the synthetic dyes - a completely xenobiotic compound - as the key cofactor in respiration, substituting quinones in the electron transport chain. <br>  
Our <i>X. coli </i> will therefore only be able to survive in the presence of azodyes, a particular environment only found in the wastewater of the textile indutry that it is aimed to degrade. The biosafety strategy is embedded into the system, and tighly linked to the survival of the xenobiological organism.
Our <i>X. coli </i> will therefore only be able to survive in the presence of azodyes, a particular environment only found in the wastewater of the textile indutry that it is aimed to degrade. The biosafety strategy is embedded into the system, and tighly linked to the survival of the xenobiological organism.
-
        </p>
+
</p>
-
        </div>
+
-
<div class="textArena">
+
<br/>
 +
</div>
-
 
+
<!--- This is the second biobrick --->
-
<h3> 1. Choosing an essential cofactor </h3>
+
<div id="view3"><div class="textTitle"><h4>Choosing an Essential Co-Factor</h4></div><br>
<p>In order to create a xenobiological organism with a metabolic firewall we decided to try to design cofactor that would be essential to E-Coli metabolism that could be derived from our Azo Dye waste products. This cofactor would need to be functionally similar to an existing molecule in the E-Coli metabolism.
<p>In order to create a xenobiological organism with a metabolic firewall we decided to try to design cofactor that would be essential to E-Coli metabolism that could be derived from our Azo Dye waste products. This cofactor would need to be functionally similar to an existing molecule in the E-Coli metabolism.
Line 171: Line 83:
<div style="font-size:0.5em;">
<div style="font-size:0.5em;">
-
<p>Image Credits: EcoCyc Metabolic Database To see the full and interactive tree visit: http://goo.gl/bEK46y  
+
<p>Image Credits: EcoCyc Metabolic Database To see the full and interactive tree visit: http://goo.gl/bEK46y</p>
-
</p>
+
</div>
</div>
-
<br>
 
-
<p> Given the vast number of cofactors in the e-coli we needed a method to select the molecular structures closest to azo dye waste products. To solve this problem we developed a computer program that would do just that:
 
-
Many different azodyes exist – each of which gives different products after being broken down by cleaving enzymes. But how are these products related? Do they all look very similar? Or are they all very different? We're interested because we would like to take the products of one or more of the azodyes and use it to chemically synthesise a xenobiological compound that our engineered bacteria would absolutely need to continue to survive.
 
-
<br>
 
-
<center><img src="https://static.igem.org/mediawiki/2014/0/07/UCL2014-Nightskyv1.png" width="60%"></center>
 
-
<!--<img src="https://static.igem.org/mediawiki/2014/0/07/UCL2014-Nightskyv1.png" width="49%"> <img src="https://static.igem.org/mediawiki/2014/c/cd/UCL2014-Nightsky.png" width="49%">-->
 
-
<br>
+
<br/>
-
The image above is not a map of stars or galaxies, but a map of the chemical similarity space of the products of azodye breakdown. We call it the 'Azodye Night Sky'. Here the colour denotes the colour of the original azodye (except black = white), and distance is a rough measure of the similarity of two compounds.
+
 
 +
<p>Given the vast number of cofactors in the e-coli we needed a method to select the molecular structures closest to azo dye waste products. To solve this problem we developed a computer program that would do just that:
 +
Many different azodyes exist – each of which gives different products after being broken down by cleaving enzymes. But how are these products related? Do they all look very similar? Or are they all very different? We're interested because we would like to take the products of one or more of the azodyes and use it to chemically synthesise a xenobiological compound that our engineered bacteria would absolutely need to continue to survive.</p>
 +
<br/>
 +
<center><img src="https://static.igem.org/mediawiki/2014/0/07/UCL2014-Nightskyv1.png" width="45%"></center>
 +
<br/>
 +
<p>The image above is not a map of stars or galaxies, but a map of the chemical similarity space of the products of azodye breakdown. We call it the 'Azodye Night Sky'. Here the colour denotes the colour of the original azodye (except black = white), and distance is a rough measure of the similarity of two compounds.</p>
 +
<br/>
 +
<p>This image was included in our exhibition as part of our Uncolour Me Curious event.</p>
   
   
-
This image was included in our exhibition as part of our Uncolour Me Curious event.
+
<h5>How was the Azodye Night Sky generated?</h5>
   
   
-
<h4>How was the Azodye Night Sky generated?</h4>
+
<p>There exist computational chemistry tools that can analyse the similarity between two molecules. These work by first encoding each molecule of interest as a bit string "10010001100101…" where each bit represents the presence (1) or absence (0) of some substructure within the molecule. These bit strings are known as fingerprints.</p>
   
   
-
There exist computational chemistry tools that can analyse the similarity between two molecules. These work by first encoding each molecule of interest as a bit string "10010001100101…" where each bit represents the presence (1) or absence (0) of some substructure within the molecule. These bit strings are known as fingerprints.
+
<p>We can then compare molecules by taking the bitwise AND operation on the two fingerprints. This is a function that is only 1 if both molecules are 1. For example:
-
+
-
We can then compare molecules by taking the bitwise AND operation on the two fingerprints. This is a function that is only 1 if both molecules are 1. For example:
+
<pre>
<pre>
       A = 0110101...
       A = 0110101...
Line 209: Line 120:
Finally we can plot the map of our molecules – incorporating their fingerprint dissimilarity – our Azodye Night Sky!
Finally we can plot the map of our molecules – incorporating their fingerprint dissimilarity – our Azodye Night Sky!
   
   
-
This work was performed in the Python programming language using the RDKit package (to generate molecules, fingerprints, and dissimilarity).
+
This work was performed in the Python programming language using the RDKit package (to generate molecules, fingerprints, and dissimilarity).</p>
-
+
-
<h4>XenoRank: A tool for prioritising xenobiological synthesis</h4>
+
   
   
 +
</div>
 +
 +
<!--- This is the third biobrick --->
 +
<div id="view4"><div class="textTitle"><h4>XenoRank: A Tool for Prioritising Xenobiological Synthesis</h4></div><br>
 +
<a name="robPRO">
Our Azodye Night Sky is attractive, but really we want to use these techniques to help us find suitable xenobiological compounds.
Our Azodye Night Sky is attractive, but really we want to use these techniques to help us find suitable xenobiological compounds.
   
   
Line 236: Line 150:
We have published this tool on <a href="https://github.com/robjstan/xenorank">Github</a> under an MIT licence. We hope it to be useful for other iGEM teams, and the synthetic biology community in general.
We have published this tool on <a href="https://github.com/robjstan/xenorank">Github</a> under an MIT licence. We hope it to be useful for other iGEM teams, and the synthetic biology community in general.
 +
</div>
-
<h3> 2. Designing the Xeno-quinones </h3>
+
<!--- This is the fourth biobrick --->
 +
<div id="view5"><div class="textTitle"><h4>Designing the Xeno-quinones</h4></div><br>
<p> The pathway chosen from XenoRank was that of the cofactors ubiquinone and menaquinone. They are the electron buffer substituent of coenzyme Q in complex III of the electron transport chain that creates ATP on the membrane of E-Coli. </p>
<p> The pathway chosen from XenoRank was that of the cofactors ubiquinone and menaquinone. They are the electron buffer substituent of coenzyme Q in complex III of the electron transport chain that creates ATP on the membrane of E-Coli. </p>
<br>
<br>
-
<img src="https://static.igem.org/mediawiki/2014/d/d4/ETC-Graphic.png">
+
<img src="https://static.igem.org/mediawiki/2014/d/d4/ETC-Graphic.png" style="margin-left:15%">
<br>
<br>
-
<p> Ubiquinone is the electron buffer used in aerobic conditions and menaquinone in anaerobic conditions. Both quinols produce protons and electrons to create the electrochemical gradient used later in Complex IV and V to yield ATP.  
+
<p> Ubiquinone is the electron buffer used in aerobic conditions and menaquinone in anaerobic conditions. Both quinols produce protons and electrons to create the electrochemical gradient used later in Complex IV and V to yield ATP.  In order to design Xeno versions of these molecules, it was important to ensure that we understood what each constituent on the the molecule provided to the chemical reaction.  
</p>
</p>
-
<br>
+
 
-
<img src="https://static.igem.org/mediawiki/2014/4/49/ETC-Detailed-Graphic.png" width="60%">
+
<table>
-
<br>
+
  <tr>
-
<p>In order to design Xeno versions of these molecules, it was important to ensure that we understood what each constituent on the the molecule provided to the chemical reaction.
+
    <td width="50%"><img src="https://static.igem.org/mediawiki/2014/4/49/ETC-Detailed-Graphic.png" width="80%"/></td>
-
</p>
+
    <td width="50%"><img src="https://static.igem.org/mediawiki/2014/0/0d/Properties-of-Ubiquinone-and-Menaquinone.png" width="80%"/></td>
-
<br>
+
  <tr>
-
<img src="https://static.igem.org/mediawiki/2014/0/0d/Properties-of-Ubiquinone-and-Menaquinone.png" width="60%">
+
</table>
-
<br>
+
 
<p>From this understanding we designed the following molecules for our xeno-coli that we believe retains the properties described above  
<p>From this understanding we designed the following molecules for our xeno-coli that we believe retains the properties described above  
</p>
</p>
-
<br>
+
<br><br/>
-
<img src="https://static.igem.org/mediawiki/2014/1/19/Xenoquinones-Graphic.png" width="60%">
+
<img src="https://static.igem.org/mediawiki/2014/1/19/Xenoquinones-Graphic.png" width="60%" style="margin-left:22%">
-
<br>
+
<br/><br/>
<p>Once we The next step was to generate the organic chemical mechanism to go from the azo dye waste products to the new quinols. They had to be generated via organic chemistry as opposed to synthetic biology because if they could be produced by biological mechanisms they would not work as xeno molecules (please see science of xeno section).
<p>Once we The next step was to generate the organic chemical mechanism to go from the azo dye waste products to the new quinols. They had to be generated via organic chemistry as opposed to synthetic biology because if they could be produced by biological mechanisms they would not work as xeno molecules (please see science of xeno section).
</p>
</p>
<br>
<br>
-
<img src="https://static.igem.org/mediawiki/2014/8/8a/Sir-Xenoquinone-Pathway-from-Mordant-Brown-33.png" width="60%">
 
-
<img src="https://static.igem.org/mediawiki/2014/d/de/Sir-Bondiquinone-Pathway-from-Acid-Orange-7.png" width="60%">
 
-
<br>
 
-
<h3> 3. Replacing the Natural Quinones </h3>
+
<table>
 +
  <tr valign="top">
 +
    <td width="50%"><img src="https://static.igem.org/mediawiki/2014/8/8a/Sir-Xenoquinone-Pathway-from-Mordant-Brown-33.png" width="95%"/></td>
 +
    <td width="50%"><img src="https://static.igem.org/mediawiki/2014/d/de/Sir-Bondiquinone-Pathway-from-Acid-Orange-7.png" width="95%" style="margin-top:-21px;"/></td>
 +
  <tr>
 +
</table>
 +
 
 +
<h5>Replacing the Natural Quinones </h5>
<p>In order for our organism to become an autotroph to our xenobiological quinones it was necessary for us to remove the natural quinones. The best approach it seemed was to do a gene knock out for a biosynthesis step of ubiquinone and menaquinone. The metabolic pathway provided a number of options  
<p>In order for our organism to become an autotroph to our xenobiological quinones it was necessary for us to remove the natural quinones. The best approach it seemed was to do a gene knock out for a biosynthesis step of ubiquinone and menaquinone. The metabolic pathway provided a number of options  
</p>
</p>
 +
<br/>
 +
<img src="https://static.igem.org/mediawiki/2014/c/ca/UCL_2014_Pathway_tube.jpg" width="50%" style="margin-left:25%"/></td>
-
<img src =  "https://static.igem.org/mediawiki/2014/c/ca/UCL_2014_Pathway_tube.jpg" alt="metabolic tube map" style="width:25%">
+
<br/>
 +
<br/>
-
<p>A search of the literature showed that a knockout of ispB successfully killed e-coli colonies as it is <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC179075/">[1]essential for <i>E. coli's</i></a> . ispB codes for the protein that attaches the hydrophobic side chain on the quinones, allowing them to dock in the membrane. As ispB is used to synthesise all electron buffer quinones, it was the perfect knock out as it meant that only one was required.</p>  
+
<p>In order for our organism to become an autotroph to our xenobiological quinones it was necessary for us to remove the natural quinones. The best approach it seemed was to do a gene knock out for a biosynthesis step of ubiquinone and menaquinone. The metabolic pathway provided a number of options.  A search of the literature showed that a knockout of ispB successfully killed e-coli colonies as it is <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC179075/">[1]essential for <i>E. coli's</i></a> . ispB codes for the protein that attaches the hydrophobic side chain on the quinones, allowing them to dock in the membrane. As ispB is used to synthesise all electron buffer quinones, it was the perfect knock out as it meant that only one was required.</p>  
<div style="font-size:0.5em;">
<div style="font-size:0.5em;">
<p>See paper here: http://goo.gl/MlMY3c  
<p>See paper here: http://goo.gl/MlMY3c  
</p>
</p>
</div>
</div>
-
<h4>Antisense RNA gene Silencing</h4>
 
-
<p> Our first step was to try a proof of concept knockdown technique: antisense RNA gene silencing. We designed primers to obtain the reverse complement strand of a section of the gene we want to silence. When this section is transcribed it interferes with ispB affecting its translation. 
 
 +
</div>
 +
<!--- This is the fifth biobrick --->
 +
<div id="view6"><div class="textTitle"><h4>Antisense RNA Gene Silencing</h4></div><br>
 +
 +
<p> Our first step was to try a proof of concept knockdown technique: antisense RNA gene silencing. We designed primers to obtain the reverse complement strand of a section of the gene we want to silence. When this section is transcribed it interferes with ispB affecting its translation. 
</p>
</p>
<br>
<br>
Line 294: Line 220:
<p>The knockdown  <a href="https://2014.igem.org/Team:UCL/Science/Results/Xeno#Xeno">showed to be unsuccessful in reducing bacteria growth</a> and therefore cannot be use to create a xenobiological organism. The next step to try was a full knock out using the crispr technique </p>
<p>The knockdown  <a href="https://2014.igem.org/Team:UCL/Science/Results/Xeno#Xeno">showed to be unsuccessful in reducing bacteria growth</a> and therefore cannot be use to create a xenobiological organism. The next step to try was a full knock out using the crispr technique </p>
<br>
<br>
-
<h3>CRISPR Knock Outs</h3>
+
<h5>CRISPR Knock Outs</h5>
<p>The CRISPR technique is based on the bacterial ability to cut out or replace viral DNA that has been inserted into it’s plasmids. We now can now utilise the Cas9 protein with a target sequence to do cut out of any gene within a plasmid.
<p>The CRISPR technique is based on the bacterial ability to cut out or replace viral DNA that has been inserted into it’s plasmids. We now can now utilise the Cas9 protein with a target sequence to do cut out of any gene within a plasmid.
</p>
</p>
Line 308: Line 234:
</div>
</div>
<p> Despite our plans there was not sufficient time to complete a knockout using CRISPR and that will be our next step beyond the competition. </p>
<p> Despite our plans there was not sufficient time to complete a knockout using CRISPR and that will be our next step beyond the competition. </p>
 +
 +
</div>
 +
 +
<!--- This is the fifth biobrick --->
 +
<div id="view7"><div class="textTitle"><h4> References:</h4></div><br>
<div class="referenceBoring">
<div class="referenceBoring">
-
<h4> References:</h4>
 
<ol>
<ol>
     <li>Wright, O., Stan, G.-B., and Ellis, T. (2013). Building-in biosafety for synthetic biology. (Review) <em>Microbiology</em>, <strong>159</strong>, 1221-1235. <a href="http://www.ncbi.nlm.nih.gov/pubmed/23519158">http://www.ncbi.nlm.nih.gov/pubmed/23519158</a> </li>
     <li>Wright, O., Stan, G.-B., and Ellis, T. (2013). Building-in biosafety for synthetic biology. (Review) <em>Microbiology</em>, <strong>159</strong>, 1221-1235. <a href="http://www.ncbi.nlm.nih.gov/pubmed/23519158">http://www.ncbi.nlm.nih.gov/pubmed/23519158</a> </li>
Line 318: Line 248:
   <li>Malyshev, D.A., Dhami, K., Lavergne, T. et al. (2014). A semi-synthetic organism with an expanded genetic alphabet <em>Nature</em>, <strong>509</strong>, 385-388. <a href="http://www.nature.com/nature/journal/v509/n7500/full/nature13314.html">http://www.nature.com/nature/journal/v509/n7500/full/nature13314.html </a></li>  
   <li>Malyshev, D.A., Dhami, K., Lavergne, T. et al. (2014). A semi-synthetic organism with an expanded genetic alphabet <em>Nature</em>, <strong>509</strong>, 385-388. <a href="http://www.nature.com/nature/journal/v509/n7500/full/nature13314.html">http://www.nature.com/nature/journal/v509/n7500/full/nature13314.html </a></li>  
</ol><br/>
</ol><br/>
 +
</div>
 +
 +
</div>
 +
</div>
</div>
Line 343: Line 277:
.textArena {
.textArena {
   background-color: white;
   background-color: white;
-
   padding: 0% 8% 0% 8%;
+
   padding: 8% 8% 8% 8%;
   font-size: 90%;
   font-size: 90%;
   font-family: 'Open Sans', 'Helvetica Neue', Helvetica, Arial, sans-serif;
   font-family: 'Open Sans', 'Helvetica Neue', Helvetica, Arial, sans-serif;
Line 404: Line 338:
.inlinegb {
.inlinegb {
display:inline-block;
display:inline-block;
 +
}
 +
 +
div.SCJBBHIGHLIGHT p {
 +
  color: #fff;
 +
  border: solid 4px #F7931E;
 +
  border-radius: 10px;
 +
  background-color: #000;
 +
  display: table;
 +
  padding: 1%;
 +
  margin: 1px 1px 0px 1px;
 +
  box-shadow: 10px 10px 5px #888888;
}
}
Line 470: Line 415:
/*==DesignXeno==*/
/*==DesignXeno==*/
 +
    .designXenoBig{
 +
    width:100%;
 +
 +
 +
    }
     .designXeno {
     .designXeno {
     height:280px;
     height:280px;
Line 475: Line 425:
     background-color:#BFD9E5;
     background-color:#BFD9E5;
     }
     }
 +
/*========Biological vs. Xenobiological strategies========*/
/*========Biological vs. Xenobiological strategies========*/

Latest revision as of 02:35, 18 October 2014

Goodbye Azodye UCL iGEM 2014

Xenobiology

Xenobiology - The Ultimate Biosafety Tool


The wide use of genetically modified organisms causes concerns on how they will interact in the natural environment. In particular could the genetically modified microbes escape our constrains, and outcompete the organisms found in the natural ecosystem? Could the DNA we inserted into a specific bacteria be transmitted, with unknown spread of information?
Since the early days of genetic engineering we had to reflect on biosafety strategies to control these risks, and synthetic biology is bringing these concerns to another level: the more we tinker with biology, the more our biosafety needs to be bullet-proof.

Xenobiology implements the term "synthetic" by creating organisms that are unable to survive in the natural environment and necessitate an artificial intervention from man to exist. It aims to create a synthetic "man-made" version of Biology, that respects the definition of life, but is based on entirely different mechanisms to function. The biochemistry of a xeno-organism uses new XNAs, genetic codes and cofactors different from the ones explored by Biology and is therefore incompatible with other forms of life. This allows a much higher level of control: a xeno-organism will not be able to find the xenocompounds in the natural environmentnor will be able to use bacterial communication systems.



We explored this possibility with the longer term vision of creating an X. coli which is metabolically dependent on azo dyes. An alien form of life, different from the one we know, will merge synthetic chemistry with synthetic biology - allowing the remediate the damage that the first one caused and making the remediating agent dependent on the toxic compounds. This system would be completely incompatible and invisible to regular biology, now we can ask: is non-biological life safe enough?


Biological vs. Xenobiological Strategies


Biosafety strategies have so far explored biology to implement clever control mechanisms to control. They investigated various strategies that allow to kill bacteria when needed or that hinder genetic information to spread among different organisms. Our biosafety strategy is exploring the regions outside of Biology, with the ultimate goal of bringing Biology to a parallel domain where it does not interact with our own one. Why tinkering with our same Biology when we can create a new on, at the same time biology and technology, that we can control at a much higher level?

Biological Strategies

The biosafety mechanism is added to the system as additional layers of protection, the most explored are:

  1. Restriction enzyme systems: autodestruction of the transformed plasmid when task ended
  2. Semantic containment: different meaning of stop codon, other bacteria will read as stop e.g. amber codon
  3. Auxotrophy: knock-out of biosynthesis of a key naturally produced compound that needs to be provided in the media
  4. Suicide system: bacteria die when finished its task/changes environment e.g. toxin/antitoxin where the bacteria stops producing antitoxin when triggered hence dies
Xenobiological Strategies

The safety mechanism embedded is into the system on three different levels:

  1. Genetic Firewall: Use of XNAs, incompatible with other organisms and synthetic nucleotides not found in nature
  2. Semantic Firewall: Genetic code has a different meaning than the natural code, all the codons code for a different amino acid from the standard table and could code for non-natural amino acids as well
  3. Metabolic Firewall: A Synthetic auxotrophy that uses a xenobiotic compound as key cofactor/amino acid which the bacteria is unable to produce or find in the natural enviroment
Designing Xeno-Coli

We aim to engineer the bacteria to utilise the synthetic dyes - a completely xenobiotic compound - as the key cofactor in respiration, substituting quinones in the electron transport chain.
Our X. coli will therefore only be able to survive in the presence of azodyes, a particular environment only found in the wastewater of the textile indutry that it is aimed to degrade. The biosafety strategy is embedded into the system, and tighly linked to the survival of the xenobiological organism.


Choosing an Essential Co-Factor


In order to create a xenobiological organism with a metabolic firewall we decided to try to design cofactor that would be essential to E-Coli metabolism that could be derived from our Azo Dye waste products. This cofactor would need to be functionally similar to an existing molecule in the E-Coli metabolism.

Image Credits: EcoCyc Metabolic Database To see the full and interactive tree visit: http://goo.gl/bEK46y


Given the vast number of cofactors in the e-coli we needed a method to select the molecular structures closest to azo dye waste products. To solve this problem we developed a computer program that would do just that: Many different azodyes exist – each of which gives different products after being broken down by cleaving enzymes. But how are these products related? Do they all look very similar? Or are they all very different? We're interested because we would like to take the products of one or more of the azodyes and use it to chemically synthesise a xenobiological compound that our engineered bacteria would absolutely need to continue to survive.



The image above is not a map of stars or galaxies, but a map of the chemical similarity space of the products of azodye breakdown. We call it the 'Azodye Night Sky'. Here the colour denotes the colour of the original azodye (except black = white), and distance is a rough measure of the similarity of two compounds.


This image was included in our exhibition as part of our Uncolour Me Curious event.

How was the Azodye Night Sky generated?

There exist computational chemistry tools that can analyse the similarity between two molecules. These work by first encoding each molecule of interest as a bit string "10010001100101…" where each bit represents the presence (1) or absence (0) of some substructure within the molecule. These bit strings are known as fingerprints.

We can then compare molecules by taking the bitwise AND operation on the two fingerprints. This is a function that is only 1 if both molecules are 1. For example:

      A = 0110101...
      B = 0011111...
A AND B = 0010101…
Then we can get the similarity between the two molecules by the fraction: (Number of 1s in A AND B) / (Total number of bits) But if we want to visualise this we don't actually want the similarity but instead the dissimilarity (the distance between two molecules in similarity space): dissimilarity = 1 - similarity Now imagine we have N molecules. Then the NxN dissimilarity matrix gives us the dissimilarity between any two of those molecules. But because similarity space is so complex, if we wanted to draw the map of these distances we would need to use (in general) N-1 dimensions! Because we want to draw this information in 2 dimensions, we need to use a method to reduce the number of dimensions while keeping as much of the distance information as we can. Here we have chosen to use Multidimensional Scaling (MDS). Finally we can plot the map of our molecules – incorporating their fingerprint dissimilarity – our Azodye Night Sky! This work was performed in the Python programming language using the RDKit package (to generate molecules, fingerprints, and dissimilarity).

XenoRank: A Tool for Prioritising Xenobiological Synthesis


Our Azodye Night Sky is attractive, but really we want to use these techniques to help us find suitable xenobiological compounds. So we have developed a web application to help us prioritise which azodye breakdown products are most similar to a list of xenobiological cofactor compounds that we are interested in. We've called this tool XenoRank.

We start by entering a list of molecules in the
SMILES format. These are compared with a set of default compounds of xenobiological interest. Currently this is a list of cofactor compounds.

The results of the application is a report, where the compounds we are interested in (for us Azodye breakdown products) are ordered with respect to the highest similarity to any of our xenobiological compounds.

We show the above diagram for each compound, showing the similarity to each of the xenobiological compounds. We have published this tool on Github under an MIT licence. We hope it to be useful for other iGEM teams, and the synthetic biology community in general.

Designing the Xeno-quinones


The pathway chosen from XenoRank was that of the cofactors ubiquinone and menaquinone. They are the electron buffer substituent of coenzyme Q in complex III of the electron transport chain that creates ATP on the membrane of E-Coli.



Ubiquinone is the electron buffer used in aerobic conditions and menaquinone in anaerobic conditions. Both quinols produce protons and electrons to create the electrochemical gradient used later in Complex IV and V to yield ATP. In order to design Xeno versions of these molecules, it was important to ensure that we understood what each constituent on the the molecule provided to the chemical reaction.

From this understanding we designed the following molecules for our xeno-coli that we believe retains the properties described above





Once we The next step was to generate the organic chemical mechanism to go from the azo dye waste products to the new quinols. They had to be generated via organic chemistry as opposed to synthetic biology because if they could be produced by biological mechanisms they would not work as xeno molecules (please see science of xeno section).


Replacing the Natural Quinones

In order for our organism to become an autotroph to our xenobiological quinones it was necessary for us to remove the natural quinones. The best approach it seemed was to do a gene knock out for a biosynthesis step of ubiquinone and menaquinone. The metabolic pathway provided a number of options




In order for our organism to become an autotroph to our xenobiological quinones it was necessary for us to remove the natural quinones. The best approach it seemed was to do a gene knock out for a biosynthesis step of ubiquinone and menaquinone. The metabolic pathway provided a number of options. A search of the literature showed that a knockout of ispB successfully killed e-coli colonies as it is [1]essential for E. coli's . ispB codes for the protein that attaches the hydrophobic side chain on the quinones, allowing them to dock in the membrane. As ispB is used to synthesise all electron buffer quinones, it was the perfect knock out as it meant that only one was required.

See paper here: http://goo.gl/MlMY3c

Antisense RNA Gene Silencing


Our first step was to try a proof of concept knockdown technique: antisense RNA gene silencing. We designed primers to obtain the reverse complement strand of a section of the gene we want to silence. When this section is transcribed it interferes with ispB affecting its translation.


The design for the primers that would amplify the gene contained the bbk prefix and suffix. The forward primer contains suffix, reverse primer contains prefix hence sequence inserted as reverse complement into the vector



The gene was then cloned into pSB1C3 under the control of lac inducible promoter to observe effect on growth.

The knockdown showed to be unsuccessful in reducing bacteria growth and therefore cannot be use to create a xenobiological organism. The next step to try was a full knock out using the crispr technique


CRISPR Knock Outs

The CRISPR technique is based on the bacterial ability to cut out or replace viral DNA that has been inserted into it’s plasmids. We now can now utilise the Cas9 protein with a target sequence to do cut out of any gene within a plasmid.

The Cas9 protein is expressed with gRNA that is complimentary for the target sequence within the DNA you wish to cut out with a PAM (Protospacer Adjacent Motif) sequence downstream of the target. The PAM sequence in e-coli must be an NGG sequence (N representing any base)

To design our gRNA we used the target sequence online design tool DNA 2.0: https://www.dna20.com/eCommerce/cas9/input

Our gRNA target sequences were as follows:

Despite our plans there was not sufficient time to complete a knockout using CRISPR and that will be our next step beyond the competition.

References:


  1. Wright, O., Stan, G.-B., and Ellis, T. (2013). Building-in biosafety for synthetic biology. (Review) Microbiology, 159, 1221-1235. http://www.ncbi.nlm.nih.gov/pubmed/23519158
  2. Okada, K., Minehira, M., and Zhu, X. (1997). The ispB gene encoding octaprenyl diphosphate synthase is essential for growth of Escherichia coli. Journal of Bacteriology, 179, 3058–3060. http://www.ncbi.nlm.nih.gov/pubmed/9139929
  3. Søballe, B. , Poole, K. R. (1999). Microbial ubiquinones: multiple roles in respiration, gene regulation and oxidative stress management. (Review) Microbiology, 145, 1817-1830. http://www.ncbi.nlm.nih.gov/pubmed/10463148
  4. Schmidt, M (2010). Xenobiology: A new form of life as the ultimate biosafety tool Bioessays, 32, 322-331. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2909387/
  5. Malyshev, D.A., Dhami, K., Lavergne, T. et al. (2014). A semi-synthetic organism with an expanded genetic alphabet Nature, 509, 385-388. http://www.nature.com/nature/journal/v509/n7500/full/nature13314.html

Contact Us

University College London
Gower Street - London
WC1E 6BT
Biochemical Engineering Department
Phone: +44 (0)20 7679 2000
Email: ucligem2014@gmail.com

Follow Us