Team:UCL/Science/Proto
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<!-- Titles go in a <h1>TITLE GOES HERE</h1> and h1 is this biggest title and h6 is the smallest. all paragraphs go in <p>paragraph goes here</p> tags. Images go in as <img src="url of image here"> and to upload an image go to https://2014.igem.org/Special:Upload. Upload the image then click on the image which takes you to a page with only an image on it. The url of the image is the image you want to use. Use google and ask Lewis and Adam as much as you want--> | <!-- Titles go in a <h1>TITLE GOES HERE</h1> and h1 is this biggest title and h6 is the smallest. all paragraphs go in <p>paragraph goes here</p> tags. Images go in as <img src="url of image here"> and to upload an image go to https://2014.igem.org/Special:Upload. Upload the image then click on the image which takes you to a page with only an image on it. The url of the image is the image you want to use. Use google and ask Lewis and Adam as much as you want--> | ||
- | < | + | <!--- This is the coding for the tabs (ask sanjay before altering this) ---> |
- | < | + | <ul class="tabs"> |
+ | <li><a href="#view1">UCL iGEM 2014</a></li> | ||
+ | <li><a href="#view2">Azoreductase</a></li> | ||
+ | <li><a href="#view3">Laccase</a></li> | ||
+ | <li><a href="#view4">Lignin Peroxidase</a></li> | ||
+ | <li><a href="#view5">Bacterial Peroxidases</a></li> | ||
+ | <li><a href="#view6">ispB RNAi</a></li> | ||
+ | <li><a href="#view7">Nuclease</a></li> | ||
+ | </ul> | ||
+ | <div class="tabcontents"> | ||
- | < | + | <!--- This is the overview section ---> |
- | + | <div id="view1"><div class="textTitle"><h4>Our BioBricks & how they lead to azo degradation</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> | ||
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<!-- =========================STOP========================== --> | <!-- =========================STOP========================== --> |
Revision as of 17:03, 16 October 2014
Our BioBricks & how they lead to azo degradation
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 (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.
Spore Coat Protein Laccase (BBa_K1336002)
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.
Laccase (BBa_K729006)
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.
Lignin Peroxidase (BBa_K500000)
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.
Bacillus subtilis dye-decolorizing peroxidase (BsDyP) (BBa_K1336003)
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.
Pseudomonas putida MET94 dye-decolorizing peroxidase (PpDyP) (BBa_K1336004)
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.
Octaprenyl Diphosphate Synthase (ispB) (BBa_K1336005)
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.
Extracellular Nuclease (nucB) (BBa_K729004)
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.