Team:UCL/Project/Biobricks

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<div class="textTitle"><h4>Overview</h4></div><br>
<div class="textTitle"><h4>Overview</h4></div><br>
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<p data-step="1" data-position='top' data-intro="This is the overview of our BioBricks, how they work together and our approach.">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="Click here to learn more about our bioprocess!" 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>
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<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="Click here to learn more about our bioprocess!" 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>
<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>
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<a data-tip="true" class="top large" data-tip-content="This diagram explains the basic construct of a 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>
<a data-tip="true" class="top large" data-tip-content="This diagram explains the basic construct of a 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>
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<p data-step="2" data-position='top' data-intro="Azoreductase will be used to cleave the azo bond.">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>
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<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>
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<div id="view3"><div class="textTitle"><h4>Azoreductase 1B6 (BBa_K1336001)</h4></div><br>
<div id="view3"><div class="textTitle"><h4>Azoreductase 1B6 (BBa_K1336001)</h4></div><br>
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<p data-step="3" data-position='top' data-intro="We chose to study 2 azoreductases, allowing us to determine the best option via characterisation studies.">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>
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<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>
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<div id="view4"><div class="textTitle"><h4><a name="BBa_K1336003"><span>Lignin Peroxidase (BBa_K1336003)</span></a></h4></div><br>
<div id="view4"><div class="textTitle"><h4><a name="BBa_K1336003"><span>Lignin Peroxidase (BBa_K1336003)</span></a></h4></div><br>
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<p data-step="4" data-position='top' data-intro="Lignin peroxidase allows the oxidation of the amines produced from the azo bond cleavage reaction.">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>
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<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>
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<a data-tip="true" class="top large" data-tip-content="Here's our BsDyP 'chilling out' before PCR." href="javascript:void(0)" style="width: 30%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/8/89/UCLicebucketlabSCJ.JPG" style="max-width: 100%;"></a>
<a data-tip="true" class="top large" data-tip-content="Here's our BsDyP 'chilling out' before PCR." href="javascript:void(0)" style="width: 30%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/8/89/UCLicebucketlabSCJ.JPG" style="max-width: 100%;"></a>
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<p data-step="5" data-position='top' data-intro="BsDyP is a novel study we will be undertaking.">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><br><br>
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<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><br><br>
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<div id="view6"><div class="textTitle"><h4><a name="BBa_K1336005"><span><em>Pseudomonas putida</em> MET94 dye-decolorizing peroxidase (PpDyP) (BBa_K1336005)</span></a></h4></div><br>
<div id="view6"><div class="textTitle"><h4><a name="BBa_K1336005"><span><em>Pseudomonas putida</em> MET94 dye-decolorizing peroxidase (PpDyP) (BBa_K1336005)</span></a></h4></div><br>
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<p data-step="6" data-position='top' data-intro="PpDyP is interesting because it could potentially allow our system to degrade other compounds.">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>
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<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>
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Revision as of 13:37, 15 October 2014

Goodbye Azodye UCL iGEM 2014

BioBricks

Overview


We plan to create a complete synthetic azo dye decolourising device in E. coli 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 E. coli device and design a bioprocess which could be used to upscale the method to an industrial context.


In an industrial setting, these enzymes would work sequentially in a bioreactor with preset dynamic conditions. First, azoreductase will cleave the azo-bond (N=N), 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 toxicity levels.

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 algal-bacterial symbiosis.


Azoreductase R2 (BBa_K1336000)


This non-specific enzyme was isolated from Bacillus subtilis, although it is also found in other bacterial species. It starts the degradation of azo dyes by cleaving the azo bond.

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 Sulfasalazine. We will isolate this enzyme from B. subtilis and convert it to BioBrick format via polymerase chain reaction (PCR).



Azoreductase 1B6 (BBa_K1336001)


Another azoreductase that we will be using is isolated from Pseudomonas aeruginosa. 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.

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.


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 azo dye degradation and decolourisation.

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.


Found in B. subtilis, 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)).

The BioBrick will be constructed via PCR.






This enzyme is found in P. putida. 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 variety of substrates very efficiently. This will broaden the spectrum of action of our decolourising device, and thus being able to degrade other toxic compounds typically found in industrial wastewaters.

This BioBrick will be constructed via PCR.

Contact Us

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

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