http://2014.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=100&target=Ninglu&year=&month=2014.igem.org - User contributions [en]2024-03-29T02:31:11ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:UCL/Science/ModelTeam:UCL/Science/Model2014-10-18T01:33:53Z<p>Ninglu: </p>
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<li><a href="#view1">Modelling Degradation</a></li><br />
<li><a href="#view2">Parameter Inference</a></li><br />
<li><a href="#view3">Flux Balance Analysis</a></li><br />
<li><a href="#view4">Enzyme Kinetics</a></li><br />
<li><a href="#view5">Chemical Mechanism</a></li><br />
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<h4>Overview</h4><br />
<p> There are three ways we can degrade azodyes: using Azoreductase (AzoR), Laccase (Lac) or BsDyp. Azoreductase breaks down AzoDye (AzoD) into two products Laccase breaks down AzoDye as well as the products of the reaction of Azoreductase with AzoDye. BsDyP acts on sulfonated AzoDyes (sAzoD):</p><br />
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<center><img class="imgsizecorrect" src="https://static.igem.org/mediawiki/2014/5/51/Miriam_Pathway_v3_copy.png" width="60%"></center><br />
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<p> In order to model this system we used COPASI. We included equations for gene expression and degradation for each gene in our pathway, as well as the intake and excretion of AzoDyes and sulfonated AzoDyes. The equations we included as well as the parameter assigned to each one are shown below: </p><br />
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Equations for pathway model<br />
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<p>Using reasonable parameter values, the simulation showed that the AzoDye is degraded within two days (48 hours). This timeframe agrees with the experimental results!</p><br />
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Simulated timecourse data of Acid Orange AzoDye degradation by Azoreductase, Laccase and BsDyP<br />
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<h4>Parameter Inference</h4><br />
<p> We wanted to see which part of the pathway is the bottleneck in degrading the AzoDyes and sulfonated AzoDyes. So we analysed the parameters of our model to see which one is the most constrained, which could give us an insight on which one to tweak experimentally in the future in order to speed up the degradation. To do that we used ABC-SysBio (Liepe, 2014) .</p> <br />
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<p> Approximate Bayesian Computation (ABC) is a method that utilises Bayesian statistics for parameter inference in synthetic biology. Given a model and data form that model, it computes the most likely parameters that could give rise to that data. We used the model and simulated data we had in order to find out which parameters are restricted in the values they can have in order to achieve that behaviour. </p><br><br />
<p> To use ABC-SysBio we had to make an SBML file describing our model and write an xml input file. The input file contains values for initial conditions of each species in our model, as well as prior distributions for each parameter. The prior distributions consist of a range of values for each parameter, from which the algorithm will sample values. The input file also contains the data from the degradation of AzoDyes and sulfonated AzoDyes over two days. </p><br />
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<p>ABC-SysBio samples a value for each parameter from the priors and using the initial conditions provided, simulates the model. The resulting time course is compared to the data provided, and if the distance between the two is greater than a threshold, the sampled parameter set is rejected. This is repeated for 100 sets of samples, consisting of one population. The sets that were accepted are then perturbed by a small amount and then a new population is sampled from the perturbed sets. This process is repeated until the distance between the data and the simulations is minimised:<br />
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<img src="https://static.igem.org/mediawiki/2014/8/81/Timecourse.jpg" class="imgsizecorrect"><br />
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The parameter values that gave rise to this final population are called the 'posterior distribution'.</p> <br><br />
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Posterior distribution of model parameters<br />
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<p>The distribution of values for each parameter are shown in the diagonal. All distributions are between 0 and 1. Drawing a straight line from one parameter to the other, at the point where the two meet, the two parameters have been plotted against each other in a density contour plot. Three parameters stand out as very constricted, k3, k8, k17 and k18. These are the parameters of the reactions for intake (k3) and secretion (k8) of AzoDyes as well as the intake (k17) and secretion (k18) of sulfonated AzoDyes by the cell. This shows that the bottleneck happens at those points in our pathway. So if we increase the rate of intake and secretion of AzoDyes and sulfonated AzoDyes in our synthetic pathway, we could speed up the process of degradation! </p><br />
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<h4>Flux Balance Analysis</h4><br />
<p> In order to see whether our xenobiological approach would work we wanted to check whether lack of Ubiquinone would have an effect on the growth rate of the chassis. The literature (Okada 1997 and Soballe, 1999) suggested that Ubiquinone is essential for E.coli growth so we decided to put that to the test! In order to do that we used Flux Balance Analysis (FBA). FBA is a method that uses the metabolism model of E.coli (see below) and calculates the flow of metabolites through that system that is required to maximise a given objective.</p><br />
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The image below demonstrates the <i>E. coli</i> metabolism plotted in Cytoscape (Cline, 2007):<br />
<img src="https://static.igem.org/mediawiki/2014/a/a0/Ecoli_hairball.png" class="imgsizecorrect"><br />
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<p> In our case we used growth rate as the objective to maximise. We performed FBA for the core E.coli metabolism with and without Ubiquinone present. With Ubiquinone present the growth rate was calculated to be 0.98 h<sup> -1 </sup>. Without Ubiquione in the system the growth rate was found to be 0 h<sup> -1 </sup>, indicating that E.coli would not grow and survive without ubiquione. This suggested that silencing the essential genes for Ubiquinone production and supplying it externally would give us control over the survival of the chassis and ultimately allow us to contain it.</p><br />
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Core metabolism map used for FBA<br />
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<p> Currently Synthetic biology is primarily based on the use of active modules (usually enzymes) from organisms to create one single organism that can successfully execute a goal. However without understanding the enzymatic action on a molecular scale we are unlikely to ever be able to improve them or design our own. Be believe that this will be the future of SynBio and therefore we have made a special effort to further the understanding of the enzymes we are using via chemical mechanism modelling in conjunction with our chemistry department. </p><br />
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<h4>Azo Reductase</h4><br />
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<p>The mechanism of reductive cleavage can either be thought of a step wise addition of H+ ions and electrons or hydride and H+ ions in concert as pictured below <br />
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<h4>Laccase and Peroxidases</h4><br />
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<p>Although many papers have touched on these oxidation mechanisms; they tend to skip steps and don’t make entire sense. Examples of this exist in [1]:<br />
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<img src="https://static.igem.org/mediawiki/2014/d/df/Screen_Shot_2014-10-17_at_18.43.08.png"><br />
<p>It’s issues include radicals gaining electrons and remaining radicals. Protons disappearing and more of the like. We have therefore worked hard to create a mechanism that makes sense.<br />
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<img src="https://static.igem.org/mediawiki/2014/0/06/Oxidising_Azo_Pathway_General.png" width="90%"><br />
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<p> Other reactions such as the polymerisation are lacking literature completely and therefore have been modelled as below. The example polymerisation is via the azo reductase product of mordant brown 33. </p><br />
<img src="https://static.igem.org/mediawiki/2014/1/11/Polymerisationreactionlaccase.png" width="90%"><br />
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<h4> References </h4><br />
<p> [1] Phenolic Azo Dye Oxidation by Laccase from Pyricularia oryzae, Appl. Environ. Microbiol.December 1995 vol. 61 no. 124374-4377</p><br />
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<p>Enzyme kinetics are used to further understand reaction parameters of the enzyme. Enzyme kinetics are largely based on the Michaelis-Menten kinetic model that allows us to calculate Vmax (The maximum rate of reaction) and Km (Michaelis Constant: the substrate concentration at which the reaction rate is at half-maximum).<br />
<img src="https://static.igem.org/mediawiki/2014/0/01/Screen_Shot_2014-10-18_at_01.26.02.png"><br />
<p> Where [S]=Substrate concentration and V=Rate of Reaction<br />
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<p>The lineweaver burke plot is a double reciprocal plot of 1/[S] against 1/[V] that allows 1/Vmax and -1/Km to be understood via y and x intercepts respectively. We used our data for the decolorisation via enzyme BsDyp (see data page) to create a lineweaver burke plot and hence infer the values of Vmax and Km. <br />
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<img src=https://static.igem.org/mediawiki/2014/b/b8/Lineweaver_Burke_Plot.png><br />
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<p>From this we can infer that Vmax=0.0305 mg/mol per hour (4 d.p.) and Km=0.0034 mg/mol (4 d.p.)<br />
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<h4>References</h4><br />
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<p> Liepe, J., Kirk, P., Filippi, S., Toni, T., et al. (2014) A framework for parameter estimation and model selection from experimental data in systems biology using approximate Bayesian computation. [Online] 9 (2), 439–456.</p> <br />
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<p> Hoops S., Sahle S., Gauges R., Lee C., Pahle J., Simus N., Singhal M., Xu L., Mendes P. and Kummer U. (2006). COPASI: a COmplex PAthway SImulator. Bioinformatics 22, 3067-74.</p> <br />
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<p> Cline, M.S., Smoot, M., Cerami, E., Kuchinsky, A., et al. (2007) Integration of biological networks and gene expression data using Cytoscape. Nature Protocols. [Online] 2 (10), 2366–2382. </p><br />
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<p> Orth, J.D., Thiele, I. & Palsson, B.O. (2010) What is flux balance analysis? Nature Biotechnology. [Online] 28 (3), 245–248. </p><br />
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<p> Okada, K., Minehira, M., Zhu, X., Suzuki, K., et al. (1997) The ispB gene encoding octaprenyl diphosphate synthase is essential for growth of Escherichia coli. Journal of bacteriology. 179 (9), 3058–3060. </p><br />
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<p> Søballe, B. & Poole, R.K. (1999) Microbial ubiquinones: multiple roles in respiration, gene regulation and oxidative stress management. Microbiology (Reading, England). 145 ( Pt 8)1817–1830 </p><br />
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{{:Team:UCL/Template:footerx}}</div>Ningluhttp://2014.igem.org/Team:UCL/Project/XenobiologyTeam:UCL/Project/Xenobiology2014-10-18T01:32:03Z<p>Ninglu: </p>
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<li><a href="#view2">Strategies</a></li><br />
<li><a href="#view3">Cofactors</a></li><br />
<li><a href="#view4">XenoRank</a></li><br />
<li><a href="#view5">Xeno-quinones</a></li><br />
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<div id="view1"><div class="textTitle"><h4>Xenobiology - The Ultimate Biosafety Tool</h4></div><br><br />
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<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 />
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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 />
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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.<br />
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<p>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?<br />
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<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.<br />
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?<br />
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<h5>Biological Strategies</h5><br />
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<p>The biosafety mechanism is added to the system as additional layers of protection, the most explored are:<br />
<ol><br />
<li>Restriction enzyme systems: autodestruction of the transformed plasmid when task ended </li><br />
<li>Semantic containment: different meaning of stop codon, other bacteria will read as stop e.g. amber codon </li><br />
<li>Auxotrophy: knock-out of biosynthesis of a key naturally produced compound that needs to be provided in the media </li><br />
<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><br />
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<h5>Xenobiological Strategies</h5><br />
<p>The safety mechanism embedded is into the system on three different levels:<br />
<ol><br />
<li>Genetic Firewall: Use of XNAs, incompatible with other organisms and synthetic nucleotides not found in nature</li><br />
<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><br />
<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><br />
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<h5>Designing Xeno-Coli</h5><br />
<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> <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.<br />
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<div id="view3"><div class="textTitle"><h4>Choosing an Essential Co-Factor</h4></div><br><br />
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<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.<br />
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<img src="https://static.igem.org/mediawiki/2014/0/01/UCLEcolimetabolictree.png" width="85%"><br />
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<p>Image Credits: EcoCyc Metabolic Database To see the full and interactive tree visit: http://goo.gl/bEK46y</p><br />
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<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:<br />
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 />
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<center><img src="https://static.igem.org/mediawiki/2014/0/07/UCL2014-Nightskyv1.png" width="45%"></center><br />
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<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 />
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<p>This image was included in our exhibition as part of our Uncolour Me Curious event.</p><br />
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<h5>How was the Azodye Night Sky generated?</h5><br />
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<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><br />
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<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:<br />
<pre><br />
A = 0110101...<br />
B = 0011111...<br />
A AND B = 0010101…<br />
</pre><br />
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Then we can get the similarity between the two molecules by the fraction:<br />
(Number of 1s in A AND B) / (Total number of bits)<br />
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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):<br />
dissimilarity = 1 - similarity<br />
<br />
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!<br />
<br />
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).<br />
<br />
Finally we can plot the map of our molecules – incorporating their fingerprint dissimilarity – our Azodye Night Sky!<br />
<br />
This work was performed in the Python programming language using the RDKit package (to generate molecules, fingerprints, and dissimilarity).</p><br />
<br />
</div><br />
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<div id="view4"><div class="textTitle"><h4>XenoRank: A Tool for Prioritising Xenobiological Synthesis</h4></div><br><br />
<a name="robPRO"><br />
Our Azodye Night Sky is attractive, but really we want to use these techniques to help us find suitable xenobiological compounds.<br />
<br />
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.<br />
<br><br />
<!--[IMAGE: XENORANK1]--><br />
<img src="https://static.igem.org/mediawiki/2014/1/13/UCL2014-Xenorank1.png" width="60%" center><br />
<br><br />
We start by entering a list of molecules in the <a href="http://en.wikipedia.org/wiki/Simplified_molecular-input_line-entry_system">SMILES</a> format. These are compared with a set of default compounds of xenobiological interest. Currently this is a list of cofactor compounds.<br />
<br />
<!--[IMAGE: XENORANK2]--><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/0/09/UCL2014-Xenorank2.png" width="60%"><br />
<br><br />
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.<br />
<br />
<!--[IMAGE: XENORANK3]--><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/2/20/UCL2014-Xenorank3.png" width="60%"> <br />
<br><br />
We show the above diagram for each compound, showing the similarity to each of the xenobiological compounds.<br />
<br />
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.<br />
<br />
</div><br />
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<!--- This is the fourth biobrick ---><br />
<div id="view5"><div class="textTitle"><h4>Designing the Xeno-quinones</h4></div><br><br />
<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><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/d/d4/ETC-Graphic.png" style="margin-left:15%"><br />
<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. 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. <br />
</p><br />
<br />
<table><br />
<tr><br />
<td width="50%"><img src="https://static.igem.org/mediawiki/2014/4/49/ETC-Detailed-Graphic.png" width="80%"/></td><br />
<td width="50%"><img src="https://static.igem.org/mediawiki/2014/0/0d/Properties-of-Ubiquinone-and-Menaquinone.png" width="80%"/></td><br />
<tr><br />
</table><br />
<br />
<p>From this understanding we designed the following molecules for our xeno-coli that we believe retains the properties described above <br />
</p><br />
<br><br/><br />
<img src="https://static.igem.org/mediawiki/2014/1/19/Xenoquinones-Graphic.png" width="60%" style="margin-left:22%"><br />
<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).<br />
</p><br />
<br><br />
<br />
<table><br />
<tr valign="top"><br />
<td width="50%"><img src="https://static.igem.org/mediawiki/2014/8/8a/Sir-Xenoquinone-Pathway-from-Mordant-Brown-33.png" width="95%"/></td><br />
<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><br />
<tr><br />
</table><br />
<br />
<h5>Replacing the Natural Quinones </h5><br />
<br />
<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 <br />
</p><br />
<br/><br />
<img src="https://static.igem.org/mediawiki/2014/c/ca/UCL_2014_Pathway_tube.jpg" width="50%" style="margin-left:25%"/></td><br />
<br />
<br/><br />
<br/><br />
<br />
<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> <br />
<div style="font-size:0.5em;"><br />
<p>See paper here: http://goo.gl/MlMY3c <br />
</p><br />
</div><br />
<br />
</div><br />
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<!--- This is the fifth biobrick ---><br />
<div id="view6"><div class="textTitle"><h4>Antisense RNA Gene Silencing</h4></div><br><br />
<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. <br />
</p><br />
<br><br />
<p> 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</p><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/9/9e/Kdprimer.jpg"><br />
<br><br />
<p>The gene was then cloned into pSB1C3 under the control of lac inducible promoter to observe effect on growth.<br />
</p><br />
<br />
<!--Insert link to results for bacterial growth--><br />
<br />
<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><br />
<h5>CRISPR Knock Outs</h5><br />
<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.<br />
</p><br />
<p>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)<br />
</p><br />
<p>To design our gRNA we used the target sequence online design tool DNA 2.0: https://www.dna20.com/eCommerce/cas9/input </p><br />
<img src="https://static.igem.org/mediawiki/2014/1/1c/GRNAdesignscreenshot.png"><br />
<p>Our gRNA target sequences were as follows:<br />
</p><br />
<div class="inlinegb"><br />
<img src="https://static.igem.org/mediawiki/2014/d/dc/Targetsequencexeno.png" style="margin-right:15px;" height="180" width="210"><br />
<img src="https://static.igem.org/mediawiki/2014/3/30/Schematictargetsequencexeno.png" style="margin-right:15px;" height="180" width="210"><br />
</div><br />
<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><br />
<br />
</div><br />
<br />
<!--- This is the fifth biobrick ---><br />
<div id="view7"><div class="textTitle"><h4> References:</h4></div><br><br />
<br />
<div class="referenceBoring"><br />
<ol><br />
<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><br />
<li>Okada, K., Minehira, M., and Zhu, X. (1997). The ispB gene encoding octaprenyl diphosphate synthase is essential for growth of Escherichia coli. <em>Journal of Bacteriology</em>, <strong>179</strong>, 3058–3060. <a href="http://www.ncbi.nlm.nih.gov/pubmed/9139929">http://www.ncbi.nlm.nih.gov/pubmed/9139929 </a></li> <br />
<li>Søballe, B. , Poole, K. R. (1999). Microbial ubiquinones: multiple roles in respiration, gene regulation and oxidative stress management. (Review) <em>Microbiology</em>, <strong>145</strong>, 1817-1830. <a href="http://www.ncbi.nlm.nih.gov/pubmed/10463148">http://www.ncbi.nlm.nih.gov/pubmed/10463148 </a></li> <br />
<li>Schmidt, M (2010). Xenobiology: A new form of life as the ultimate biosafety tool <em>Bioessays</em>, <strong>32</strong>, 322-331. <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2909387/">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2909387/ </a></li> <br />
<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> <br />
</ol><br/><br />
</div><br />
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{{:Team:UCL/Template:footerx}}</div>Ningluhttp://2014.igem.org/Team:UCL/Project/XenobiologyTeam:UCL/Project/Xenobiology2014-10-18T01:28:09Z<p>Ninglu: </p>
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<div class="textArena"><br />
<!--- This is the coding for the tabs (ask sanjay before altering this) ---><br />
<br />
<ul class="tabs"><br />
<li><a href="#view1">Introduction</a></li><br />
<li><a href="#view2">Strategies</a></li><br />
<li><a href="#view3">Cofactors</a></li><br />
<li><a href="#view4">XenoRank</a></li><br />
<li><a href="#view5">Xeno-quinones</a></li><br />
<li><a href="#view6">Silencing</a></li><br />
<li><a href="#view7">References</a></li><br />
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<!--- This is the overview section ---><br />
<div id="view1"><div class="textTitle"><h4>Xenobiology - The Ultimate Biosafety Tool</h4></div><br><br />
<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?<br />
<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.<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.<br />
</p><br />
<br/><br/><br />
<div class="SCJBBHIGHLIGHT"><br />
<p>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?<br />
</p></div><br />
<br />
<br />
<br />
<br/><br />
<br />
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<br />
<!--- This is the first biobrick ---><br />
<div id="view2"><div class="textTitle"><h4>Biological vs. Xenobiological strategies</h4></div><br><br />
<br />
<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.<br />
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?<br />
</p><br />
<br />
<h5>Biological strategies</h5><br />
<br />
<p>The biosafety mechanism is added to the system as additional layers of protection, the most explored are:<br />
<ol><br />
<li>Restriction enzyme systems: autodestruction of the transformed plasmid when task ended </li><br />
<li>Semantic containment: different meaning of stop codon, other bacteria will read as stop e.g. amber codon </li><br />
<li>Auxotrophy: knock-out of biosynthesis of a key naturally produced compound that needs to be provided in the media </li><br />
<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><br />
</ol><br />
<br />
<h5>Xenobiological strategies</h5><br />
<p>The safety mechanism embedded is into the system on three different levels:<br />
<ol><br />
<li>Genetic Firewall: Use of XNAs, incompatible with other organisms and synthetic nucleotides not found in nature</li><br />
<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><br />
<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><br />
</ol><br />
<br />
<h5>Designing Xeno-Coli</h5><br />
<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> <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.<br />
</p><br />
<br />
<br/><br />
<br />
</div><br />
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<!--- This is the second biobrick ---><br />
<div id="view3"><div class="textTitle"><h4>Choosing an essential cofactor</h4></div><br><br />
<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.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2014/0/01/UCLEcolimetabolictree.png" width="85%"><br />
<br />
<div style="font-size:0.5em;"><br />
<p>Image Credits: EcoCyc Metabolic Database To see the full and interactive tree visit: http://goo.gl/bEK46y</p><br />
</div><br />
<br />
<br/><br />
<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:<br />
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 />
<br/><br />
<center><img src="https://static.igem.org/mediawiki/2014/0/07/UCL2014-Nightskyv1.png" width="45%"></center><br />
<br/><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 />
<br/> <br />
<p>This image was included in our exhibition as part of our Uncolour Me Curious event.</p><br />
<br />
<h5>How was the Azodye Night Sky generated?</h5><br />
<br />
<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><br />
<br />
<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:<br />
<pre><br />
A = 0110101...<br />
B = 0011111...<br />
A AND B = 0010101…<br />
</pre><br />
<br />
Then we can get the similarity between the two molecules by the fraction:<br />
(Number of 1s in A AND B) / (Total number of bits)<br />
<br />
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):<br />
dissimilarity = 1 - similarity<br />
<br />
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!<br />
<br />
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).<br />
<br />
Finally we can plot the map of our molecules – incorporating their fingerprint dissimilarity – our Azodye Night Sky!<br />
<br />
This work was performed in the Python programming language using the RDKit package (to generate molecules, fingerprints, and dissimilarity).</p><br />
<br />
</div><br />
<br />
<!--- This is the third biobrick ---><br />
<div id="view4"><div class="textTitle"><h4>XenoRank: A tool for prioritising xenobiological synthesis</h4></div><br><br />
<a name="robPRO"><br />
Our Azodye Night Sky is attractive, but really we want to use these techniques to help us find suitable xenobiological compounds.<br />
<br />
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.<br />
<br><br />
<!--[IMAGE: XENORANK1]--><br />
<img src="https://static.igem.org/mediawiki/2014/1/13/UCL2014-Xenorank1.png" width="60%" center><br />
<br><br />
We start by entering a list of molecules in the <a href="http://en.wikipedia.org/wiki/Simplified_molecular-input_line-entry_system">SMILES</a> format. These are compared with a set of default compounds of xenobiological interest. Currently this is a list of cofactor compounds.<br />
<br />
<!--[IMAGE: XENORANK2]--><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/0/09/UCL2014-Xenorank2.png" width="60%"><br />
<br><br />
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.<br />
<br />
<!--[IMAGE: XENORANK3]--><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/2/20/UCL2014-Xenorank3.png" width="60%"> <br />
<br><br />
We show the above diagram for each compound, showing the similarity to each of the xenobiological compounds.<br />
<br />
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.<br />
<br />
</div><br />
<br />
<!--- This is the fourth biobrick ---><br />
<div id="view5"><div class="textTitle"><h4>Designing the Xeno-quinones</h4></div><br><br />
<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><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/d/d4/ETC-Graphic.png" style="margin-left:15%"><br />
<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. 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. <br />
</p><br />
<br />
<table><br />
<tr><br />
<td width="50%"><img src="https://static.igem.org/mediawiki/2014/4/49/ETC-Detailed-Graphic.png" width="80%"/></td><br />
<td width="50%"><img src="https://static.igem.org/mediawiki/2014/0/0d/Properties-of-Ubiquinone-and-Menaquinone.png" width="80%"/></td><br />
<tr><br />
</table><br />
<br />
<p>From this understanding we designed the following molecules for our xeno-coli that we believe retains the properties described above <br />
</p><br />
<br><br/><br />
<img src="https://static.igem.org/mediawiki/2014/1/19/Xenoquinones-Graphic.png" width="60%" style="margin-left:22%"><br />
<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).<br />
</p><br />
<br><br />
<br />
<table><br />
<tr valign="top"><br />
<td width="50%"><img src="https://static.igem.org/mediawiki/2014/8/8a/Sir-Xenoquinone-Pathway-from-Mordant-Brown-33.png" width="95%"/></td><br />
<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><br />
<tr><br />
</table><br />
<br />
<h5>Replacing the Natural Quinones </h5><br />
<br />
<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 <br />
</p><br />
<br/><br />
<img src="https://static.igem.org/mediawiki/2014/c/ca/UCL_2014_Pathway_tube.jpg" width="50%" style="margin-left:25%"/></td><br />
<br />
<br/><br />
<br/><br />
<br />
<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> <br />
<div style="font-size:0.5em;"><br />
<p>See paper here: http://goo.gl/MlMY3c <br />
</p><br />
</div><br />
<br />
</div><br />
<br />
<!--- This is the fifth biobrick ---><br />
<div id="view6"><div class="textTitle"><h4>Antisense RNA gene Silencing</h4></div><br><br />
<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. <br />
</p><br />
<br><br />
<p> 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</p><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/9/9e/Kdprimer.jpg"><br />
<br><br />
<p>The gene was then cloned into pSB1C3 under the control of lac inducible promoter to observe effect on growth.<br />
</p><br />
<br />
<!--Insert link to results for bacterial growth--><br />
<br />
<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><br />
<h5>CRISPR Knock Outs</h5><br />
<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.<br />
</p><br />
<p>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)<br />
</p><br />
<p>To design our gRNA we used the target sequence online design tool DNA 2.0: https://www.dna20.com/eCommerce/cas9/input </p><br />
<img src="https://static.igem.org/mediawiki/2014/1/1c/GRNAdesignscreenshot.png"><br />
<p>Our gRNA target sequences were as follows:<br />
</p><br />
<div class="inlinegb"><br />
<img src="https://static.igem.org/mediawiki/2014/d/dc/Targetsequencexeno.png" style="margin-right:15px;" height="180" width="210"><br />
<img src="https://static.igem.org/mediawiki/2014/3/30/Schematictargetsequencexeno.png" style="margin-right:15px;" height="180" width="210"><br />
</div><br />
<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><br />
<br />
</div><br />
<br />
<!--- This is the fifth biobrick ---><br />
<div id="view7"><div class="textTitle"><h4> References:</h4></div><br><br />
<br />
<div class="referenceBoring"><br />
<ol><br />
<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><br />
<li>Okada, K., Minehira, M., and Zhu, X. (1997). The ispB gene encoding octaprenyl diphosphate synthase is essential for growth of Escherichia coli. <em>Journal of Bacteriology</em>, <strong>179</strong>, 3058–3060. <a href="http://www.ncbi.nlm.nih.gov/pubmed/9139929">http://www.ncbi.nlm.nih.gov/pubmed/9139929 </a></li> <br />
<li>Søballe, B. , Poole, K. R. (1999). Microbial ubiquinones: multiple roles in respiration, gene regulation and oxidative stress management. (Review) <em>Microbiology</em>, <strong>145</strong>, 1817-1830. <a href="http://www.ncbi.nlm.nih.gov/pubmed/10463148">http://www.ncbi.nlm.nih.gov/pubmed/10463148 </a></li> <br />
<li>Schmidt, M (2010). Xenobiology: A new form of life as the ultimate biosafety tool <em>Bioessays</em>, <strong>32</strong>, 322-331. <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2909387/">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2909387/ </a></li> <br />
<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> <br />
</ol><br/><br />
</div><br />
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{{:Team:UCL/Template:footerx}}</div>Ningluhttp://2014.igem.org/Team:UCL/Science/ExperimentTeam:UCL/Science/Experiment2014-10-18T01:24:19Z<p>Ninglu: </p>
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<img src="https://static.igem.org/mediawiki/2014/2/21/OExperiments_Bannero.jpg" width="100%" height="100%" alt="Experiments" /><br />
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<!--- This is the coding for the tabs (ask sanjay before altering this) ---><br />
<ul class="tabs"><br />
<li><a href="#view1">Stage 1</a></li><br />
<li><a href="#view2">Stage 2</a></li><br />
<li><a href="#view3">Stage 3</a></li><br />
<li><a href="#view4">Stage 4</a></li><br />
<li><a href="#view5">Stage 5</a></li><br />
<li><a href="#view6">Stage 6</a></li><br />
<li><a href="#view7">Stage 7</a></li><br />
<li><a href="#view8">Stage 8</a></li><br />
</ul><br />
<div class="tabcontents"><br />
<br />
<!--- This is the overview section ---><br />
<div id="view1"><div class="textTitle"><h4>Stage 01: Extraction of Useful BioBrick Plasmids from iGEM 2014 Distribution Kit</h4></div><br><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<div><strong>Protocols&nbsp;&nbsp;</strong><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">competent cells</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">transformation</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">miniprep</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">digest</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">gel</span></a></div><br />
<p>We began our project by identifying a range of BioBrick parts present in the iGEM 2014 distribution kit which we required as part of our cloning strategy. These parts primarily consisted of both constituitive and inducible promoter systems with ribosome binding sites which we could then use in conjunction with our azo-reductase BioBricks to assemble a functional azo dye degrading gene. We also decided that we would use the Red Florescent Protein expressing BioBrick as a control for any further transformation experiments. As the level of DNA present within each plate of the distribution kit is insufficient to perform digest and ligation reactions on it was necessary to transform each of these plasmids into our NEB5alpha competent cells. After growing our transformed cells overnight we then mini-prepped each of them to obtain BioBrick plasmids at suitable concentrations for future experiments.</p><br><br />
<font size="2"><br />
<table border="1px" width="100%" height="auto"><br />
<thead><br />
<tr><br />
<th> </th><br />
<th> Registry ID </th><br />
<th> Name / Function </th><br />
<th> Antibiotic Resistance </th><br />
<th> Source </th><br />
<th> Size </th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<td> <center>U</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K314103">BBa_K314103</a> </td><br />
<td> &nbsp;IPTG-inducible LacI Expression Cassette </td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 1, Well 4D.</td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K314103">1638 bp</a> </td><br />
</tr><br />
<tr><br />
<td> <center>T</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a> </td><br />
<td> &nbsp;RFP Coding Device </td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 4, Well 4B. </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_J04450">1069 bp</a> </td><br />
</tr><br />
<tr><br />
<td> <center>T</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_R0010">BBa_R0010</a> </td><br />
<td> &nbsp;IPTG-inducible LacI Promoter</td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 3, Well 4G.</td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_R0010">200 bp</a> </td><br />
</tr><br />
<tr><br />
<td> <center>T</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> </td><br />
<td> &nbsp;Ribosomal Binding Site (RBS)</td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 4, Well 1N.</td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_B0034">12 bp</a> </td><br />
</tr><br />
<tr><br />
<td> <center>T</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K518012">BBa_K518012</a> </td><br />
<td> &nbsp;RBS + RFP + Double Terminator</td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 1, Well 18C.</td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K518012">828 bp</a> </td><br />
</tr><br />
<tr><br />
<td> <center>N</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K206000">BBa_K206000</a> </td><br />
<td> &nbsp;pBAD Strong Promoter</td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 3, Well 14A.</td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K206000">130 bp</a> </td><br />
</tr><br />
<tr><br />
<td> <center>! N</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_R0011">BBa_R0011</a> </td><br />
<td> &nbsp;LacI-Regulated, Lambda pL Hybrid Promoter</td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 2, Well 6D.</td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_R0011">55 bp</a> </td><br />
</tr><br />
<tr><br />
<td> <center>! N</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_B0012">BBa_B0012</a> </td><br />
<td> &nbsp;Transcription Terminator for E. coli RNA Polymerase</td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 2, Well 2B.</td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_B0012">41 bp</a> </td><br />
</tr><br />
</tbody><br />
</table><br />
</font><br />
<div><font size="1">Note: U = Used in experiments; T = Used for testing purposes but not for making BioBrick Devices; N = Transformed from Distribution Kits, but not used in experiments; ! = Problematic parts (see Parts Registry), were not used.</font></div><br />
<br />
</div><br />
<br />
<!--- This is the first biobrick ---><br />
<div id="view2"><div class="textTitle"><h4>Stage 02: Identification of Useful Genes for Making New BioBricks</h4></div><br><br />
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<strong>Identifying Azo-Dye Degrading Enzymes</strong><br />
<p>Searching through the literature, we identified a number of bacterial species (including <em>Bacillus subtilis</em> and <em>Pseudomonas sp.</em>) that have proven to degrade azo dye compounds <a href="http://www.ncbi.nlm.nih.gov/pubmed/21655981">[1]</a><a href="http://www.ncbi.nlm.nih.gov/pubmed/24475252">[2]</a><a href="http://www.itqb.unl.pt/martins/index_files/JBC2002.pdf">[3]</a><a href="http://www.ncbi.nlm.nih.gov/pubmed/23820555">[4]</a>. <br><br />
We contacted the <a href="http://www.itqb.unl.pt/martins">Microbial & Enzyme Technology Lab</a> led by Dr Lígia O. Martins at the Universidade Nova de Lisboa, who are currently researching how azo dye degrading enzymes function, and they were keen to collaborate with us on our project. They agreed to send us a set of five plasmids, each containing different genes encoding azo dye degrading enzymes from both <em>B. subtilis</em> and <em>P. putida</em> (including mutated forms found to exhibit enhanced degradation activity), for us to use in our investigations (see Table below). <br><br />
</p><br><br />
<font size="2"><br />
<table border="1px" width="100%" height="auto"><br />
<thead><br />
<tr><br />
<th> Gene ID</th><br />
<th> Name / Function </th><br />
<th> Source </th><br />
<th> Size </th><br />
<th> Plasmid </th><br />
</tr><br />
</thead><br />
<tbody><br />
<!--Lisbon plasmids--><br />
<tr><br />
<td> &nbsp;<a href="http://www.ncbi.nlm.nih.gov/pubmed/21655981">pAzoR</a> </td><br />
<td> &nbsp;FMN-dependent NADH-azoreductase 1 </td><br />
<td> &nbsp;<em>Pseudomonas putida</em> </td><br />
<td> &nbsp;612 bp </td><br />
<td> &nbsp;In expression vector: pET-21a (+) (ampicillin resistant) <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[2] <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[3] </a>, <br>initially cloned between <em>NdeI</em> and <em>BamHI</em> restriction sites. </td><br />
</tr><br />
<tr><br />
<td> &nbsp;<a href="http://www.ncbi.nlm.nih.gov/pubmed/24475252">p1B6</a> </td><br />
<td> &nbsp;AzoR Heat-stable Mutant</td><br />
<td> &nbsp;<em>Pseudomonas putida</em> </td><br />
<td> &nbsp;612 bp </td><br />
<td> &nbsp;In expression vector: pET-21a (+) (ampicillin resistant) <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[2] <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[3] </a>, <br>initially cloned between <em>NdeI</em> and <em>BamHI</em> restriction sites. </td><br />
</tr><br />
<tr><br />
<td> &nbsp;<a href="http://www.itqb.unl.pt/martins/index_files/JBC2002.pdf">pCotA</a> </td><br />
<td> &nbsp;Spore Coat Protein Laccase</td><br />
<td> &nbsp;<em>Bacillus subtilis</em> </td><br />
<td> &nbsp;1542 bp </td><br />
<td> &nbsp;In expression vector: pET-21a (+) (ampicillin resistant (ampR)) <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[2] <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[3] </a>, <br>initially cloned between <strong><em>NheI</em></strong> and <em>BamHI</em> restriction sites. </td><br />
</tr><br />
<tr><br />
<td> &nbsp;<a href="http://www.ncbi.nlm.nih.gov/pubmed/23820555">pBsDyP</a> </td><br />
<td> &nbsp;Dye Decolourising Peroxidase BSU38260</td><br />
<td> &nbsp;<em>Bacillus subtilis</em> </td><br />
<td> &nbsp;1251 bp </td><br />
<td> &nbsp;In expression vector: pET-21a (+) (ampicillin resistant) <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[2] <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[3] </a>, <br>initially cloned between <em>NdeI</em> and <em>BamHI</em> restriction sites. </td><br />
</tr><br />
<tr><br />
<td> &nbsp;<a href="http://www.ncbi.nlm.nih.gov/pubmed/23820555">pPpDyP</a> </td><br />
<td> &nbsp;Dye Decolourising Peroxidase PP_3248 </td><br />
<td> &nbsp;<em>Pseudomonas putida</em> </td><br />
<td> &nbsp;864 bp </td><br />
<td> &nbsp;In expression vector: pET-21a (+) (ampicillin resistant) <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[2] <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[3] </a>, <br>initially cloned between <em>NdeI</em> and <em>BamHI</em> restriction sites. </td><br />
</tr><br />
</tbody><br />
</table><br />
</font><br />
<br><br />
<br />
<a data-tip="true" class="top large" data-tip-content="Here's the gel visualisation showing the B. subtilis genomic DNA!" href="javascript:void(0)" style="width: 20%;float: right;margin-left:1%"><img src="https://static.igem.org/mediawiki/2014/b/b3/UCL_Bsub_Genomic_Extraction.jpeg" style="max-width: 100%;"></a><br />
<strong>Extraction of <em>B. Subtilis</em> Genomic DNA</strong><br />
<div><strong>Protocols&nbsp;&nbsp;</strong><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">DNA extraction</span></a></div><br />
<p>In the meantime, Helina (in our team), was able to obtain <em>B. subtilis</em> and <em>P. aeruginosa</em> strains for us to test whether we could retrieve azo dye degrading enzymes from their genomes, specifically, the azo-reductase gene (AzoR). This would be the first step for our first azoreductase BioBrick. <br><br />
We extracted the genomic DNA from <em>B. subtilis</em> strain using a Promega Wizard Genomic DNA extraction kit so that we could subsequently amplify the azoreducatase gene (AzoR1) and create our first azoreductase BioBrick. After completing the genomic DNA extracton we ran a gel to show that we had successfully extracted the <i>B. subtilis</i> genomic DNA.</p><br />
<br><br><br />
<br />
<!-- <div class="accordion"><br />
<h4><div class="byline"><i class="icon-user"></i><strong>Extraction of Bacillus Subtilis Genomic DNA</strong></div></h4><br />
<div><br />
<div><strong>Protocols&nbsp;&nbsp;</strong><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">DNA extraction</span></a></div><br />
<p>Our literature search identified a number of bacterial species that have been proven to degrade azo dye compounds including <i>B. subtilis</i> and <i>P. aeruginosa</i>. We were able to obtain a <i>B. subtilis</i> strain for use in our project from ?. We extracted the genomic DNA from this strain using a Promega Wizard Genomic DNA extraction kit so that we could subsequently amplify the azo-reducatase gene (AzoR1) and create our first azo-reductase BioBrick. After completing the genomic DNA extracton we ran a gel to show that we had successfully extracted the <i>B. subtilis</i> genomic DNA.</p><br />
</div><br />
<h4><div class="byline"><i class="icon-user"></i> Adam Denyer, Tanel Ozdemir &nbsp;&nbsp; <i class="icon-time"></i> <abbr class="published" title="June 13, 2014">June 13, 2014</abbr></div></h4><br />
<div><br />
<p>...</p><br />
</div><br />
</div> --><br />
<br />
</div><br />
<br />
<!--- This is the second biobrick ---><br />
<div id="view3"><div class="textTitle"><h4>Stage 03: Transforming E. coli with Azo-Dye Degrading Plasmids from Lisbon</h4></div><br><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<strong>Transforming <em>E. coli</em> with Azo-Dye Degrading Plasmids</strong><br />
<div><strong>Protocols&nbsp;&nbsp;</strong><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">competent cells</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">transformation</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">miniprep</span></a></div><br />
<p>The five azo dye degrading enzymes from Lisbon arrived as the respective genes in pET-21a (+) ampicillin resistant (ampR) expression vectors/plasmids (size: 5443 bp)<a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[1]</a><a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[2]</a>. The DNA concentrations of these plasmids, however, were insufficient to perform PCR amplification, therefore we transformed each into our own <em>E. coli</em> competent cells (grown from NEB DH5&alpha; derivatives). After growing the cells overnight, we made bacterial glycerol stocks and miniprepped the cells to obtain plasmids at sufficient concentrations for further work.</p> <br />
<br><br />
<a data-tip="true" class="top large" data-tip-content="Here's the gel visualisation showing an analytical digest of the plasmids we received from Lisbon!" href="javascript:void(0)" style="width: 40%;float: right;margin-left:1%"><img src="https://static.igem.org/mediawiki/2014/0/08/UCL_23-07-2014_Analytical_Digest_Visualisation.pptx.png" style="max-width: 100%;"></a><br />
<strong>Diagnostic Digest of Azo-Dye Degrading Plasmids</strong><br />
<div><strong>Protocols&nbsp;&nbsp;</strong><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">digest</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">gel</span></a></div><br />
<p>A diagnostic digest was performed to ascertain that these pET-21a (+) plasmids contained the gene we expected. As each plasmid possessed <em>EcoRI</em> and <em>XbaI</em> restriction sites close to the genes of interest, we performed double-digests using these recognition enzymes and predicted the digest fragments. The digestion products were visualised using gel electrophoresis (see image right). </p> <br />
<br><br><br><br><br><br><br />
<br />
</div><br />
<br />
<!--- This is the third biobrick ---><br />
<div id="view4"><div class="textTitle"><h4>Stage 04: Creation of Azo-Reductase BioBrick Parts from Plasmids</h4></div><br><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<div><strong>Protocols&nbsp;&nbsp;</strong><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">digest</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">gel</span></a></div><br />
<p>After isolating our genes of interest we attempted to use PCR as a method of prefix and suffix generation to fit the BioBrick standard assembly.</p><br />
<br/><br />
<p>Achieving a successful PCR proved difficult, this may have been due to poor reagent quality. We repeated the PCR using Taq, phusion and Pfu polymerases. We took an alternative route successfully used directionless ligation to generate the prefix and suffix for 1B6. Taq polymerase eventually gave us a successful generation of prefix and suffix for BsDyP, AzoR and ispB. Unfortunately, due to time constraints we were unable to implement site directed mutagenesis on 1B6 required to remove two illegal PstI and therefore did not submitt the part to the registry.</p><br />
<br/><br />
<br />
<strong>Diagnostic Digest of Azo-Dye Degrading Plasmids</strong><br />
<div><strong>Protocols&nbsp;&nbsp;</strong></div><br />
<p>We confirmed the success of the PCR through gel visualisation, comparing PCR products with and without prefix and suffix.</p><br />
</div><br />
<br />
<!--- This is the fourth biobrick ---><br />
<div id="view5"><div class="textTitle"><h4>Stage 05: Creation of Azo-Reductase BioBrick Parts from Plasmids</h4></div><br><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>We decided to assemble both BsDyP and ispB in a LacI cassette, inducible by IPTG. <br />
Issues with inconclusive antibiotic effectivity led to major delays in construction of these composite parts. We first had to prove our antibiotics were functioning properly before making progress on our project.</p><br/><br />
<p>We confirmed the construction of our BsDyP and ispB cassettes using analytical gel digest cutting at sites E and P.</p><br />
</div><br />
<br />
<!--- This is the fifth biobrick ---><br />
<div id="view6"><div class="textTitle"><h4>Stage 06: Diagnostic Digest of Azo-Reductase BioBrick Parts</h4></div><br><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>...</p><br><br />
</div><br />
<br />
<!--- This is the fifth biobrick ---><br />
<div id="view7"><div class="textTitle"><h4>Stage 07: Assembling Azo-Reductase BioBrick Device(s)</h4></div><br><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>...</p><br><br />
<font size="2"><br />
<table border="1px" width="100%" height="auto"><br />
<thead><br />
<tr><br />
<th> </th><br />
<th> Registry ID </th><br />
<th> Gene ID</th><br />
<th> Name / Function </th><br />
<th> Source </th><br />
<th> Size </th><br />
<th> Status </th><br />
</tr><br />
</thead><br />
<tbody><br />
<!--Lisbon plasmids--><br />
<tr><br />
<td> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336000">BBa_K1336000</a> </td><br />
<td> &nbsp;AzoR </td><br />
<td> &nbsp;FMN-dependent NADH-azoreductase 1 </td><br />
<td> &nbsp;<em>Pseudomonas putida</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336000">612 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Primers">In Progress</a>]: primers designed </td><br />
</tr><br />
<tr><br />
<td> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336001">BBa_K1336001</a> </td><br />
<td> &nbsp;1B6 </td><br />
<td> &nbsp;AzoR heat-stable mutant</td><br />
<td> &nbsp;<em>Pseudomonas putida</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336001">612 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Experiments">In Progress</a>]: to remove 2 illegal PstI sites </td><br />
</tr><br />
<tr><br />
<td> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336002">BBa_K1336002</a> </td><br />
<td> &nbsp;CotA </td><br />
<td> &nbsp;Spore Coat Protein Laccase</td><br />
<td> &nbsp;<em>Bacillus subtilis</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336002">1542 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Primers">In Progress</a>]: primers designed </td><br />
</tr><br />
<tr><br />
<td> <center><img src="https://static.igem.org/mediawiki/2014/e/e0/UCL_Bronze-metal-star.jpg" width="25px"></center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336003">BBa_K1336003</a> </td><br />
<td> &nbsp;BsDyP </td><br />
<td> &nbsp;Dye Decolourising Peroxidase BSU38260</td><br />
<td> &nbsp;<em>Bacillus subtilis</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336003">1251 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Experiments">New BioBrick Part</a>]: submitted </td><br />
</tr><br />
<tr><br />
<td> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336004">BBa_K1336004</a> </td><br />
<td> &nbsp;PpDyP </td><br />
<td> &nbsp;Dye Decolourising Peroxidase PP_3248 </td><br />
<td> &nbsp;<em>Pseudomonas putida</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336004">864 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Primers">In Progress</a>]: primers designed </td><br />
</tr><br />
<tr><br />
<td> <center><img src="https://static.igem.org/mediawiki/2014/e/e0/UCL_Bronze-metal-star.jpg" width="25px"></center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336005">BBa_K1336005</a> </td><br />
<td> &nbsp;ispB RNAi </td><br />
<td> &nbsp;RNAi of Octaprenyl Diphosphate <br>Synthase fragment </td><br />
<td> &nbsp;<em>Escherichia coli, K12 strain</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336005">562 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Experiment">New BioBrick Part</a>]: submitted </td><br />
</tr><br />
<tr><br />
<td> <center><img src="https://static.igem.org/mediawiki/2014/e/e0/UCL_Bronze-metal-star.jpg" width="25px"></center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336006">BBa_K1336006</a> </td><br />
<td> &nbsp;LacIEC+ispB </td><br />
<td> &nbsp;IPTG inducible ispB RNAi </td><br />
<td> &nbsp;<em>Escherichia coli, K12 strain </em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336006">2208 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Experiment">New BioBrick Device</a>]: submitted </td><br />
</tr><br />
<tr><br />
<td> <center><img src="https://static.igem.org/mediawiki/2014/e/e0/UCL_Bronze-metal-star.jpg" width="25px"></center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336007">BBa_K1336007</a> </td><br />
<td> &nbsp;LacIEC+BsDyP </td><br />
<td> &nbsp;IPTG inducible BsDyP </td><br />
<td> &nbsp;<em>Bacillus subtilis</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336007">2895 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Experiment">New BioBrick Device</a>]: submitted </td><br />
</tr><br />
<tr><br />
<td> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K729006">BBa_K729006</a> </td><br />
<td> &nbsp;CueO </td><br />
<td> &nbsp;Laccase </td><br />
<td> &nbsp;<em>Escherichia coli </em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K729006">1612 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Experiment">In Progress</a>]: ascertaining identity </td><br />
</tr><br />
<tr><br />
<td> <center>(<img src="https://static.igem.org/mediawiki/2014/3/36/UCL_Gold-metal-star.jpg" width="25px">)</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K500000">BBa_K500000</a> </td><br />
<td> &nbsp;LiP </td><br />
<td> &nbsp;Lignin Peroxidase </td><br />
<td> &nbsp;<em>Phanerochaete chrysosporium</em> </td> <!-- <br>(White-Rot Fungi) --><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K500000">1116 bp</a> </td> <!--Check size!--><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Results">Improved Characterisation</a>]: toxicity issues in gene synthesis. <br>&nbsp;[<a href="/Team:UCL/Science/Experiment">In Progress</a>]: to subclone into pSB1C3/pSB3C5. </td><br />
</tr><br />
<tr><br />
<td> <center><img src="https://static.igem.org/mediawiki/2014/3/36/UCL_Gold-metal-star.jpg" width="25px"></center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K729004">BBa_K729004</a> </td><br />
<td> &nbsp;nucB </td><br />
<td> &nbsp;Extracellular nuclease </td><br />
<td> &nbsp;<em>Staphylococcus aureus</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K729004">561 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Results">Improved Function</a>] </td><br />
</tr><br />
</tbody><br />
</table><br />
</font><br />
</div><br />
<br />
<!--- This is the fifth biobrick ---><br />
<div id="view8"><div class="textTitle"><h4>Stage 08: Characterisation of Azo-Reductase BioBrick Devices</h4></div><br><br />
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<p>...</p><br><br />
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<h4><a name="Expt">Placeholder. Will be removed.</a></h4><br />
<strong>Protocols&nbsp;&nbsp;</strong><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">PCR</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">analytical digest</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">gel</span></a><br />
(<a href="/Team:UCL/Science/Proto"><span class="label label-warning">digest</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">ligation</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">competent cells</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">transformation</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">miniprep</span></a>)<br />
<br><br />
<p>[Insert table of Our Genes]</p><br />
<br><br />
<div class="accordion"><br />
<h4><div class="byline"><i class="icon-user"></i> Adam Denyer, Tanel Ozdemir &nbsp;&nbsp; <i class="icon-time"></i> <abbr class="published" title="June 13, 2014">June 13, 2014</abbr></div></h4><br />
<div><br />
<p>...</p><br />
</div><br />
<h4><div class="byline"><i class="icon-user"></i> Adam Denyer, Tanel Ozdemir &nbsp;&nbsp; <i class="icon-time"></i> <abbr class="published" title="June 13, 2014">June 13, 2014</abbr></div></h4><br />
<div><br />
<p>...</p><br />
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{{:Team:UCL/Template:footerx}}</div>Ningluhttp://2014.igem.org/Team:UCL/Science/ExperimentTeam:UCL/Science/Experiment2014-10-18T01:20:50Z<p>Ninglu: </p>
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<img src="https://static.igem.org/mediawiki/2014/2/21/OExperiments_Bannero.jpg" width="100%" height="100%" alt="Experiments" /><br />
</div><br />
<br />
<div class="textArena"><br />
<!--- This is the coding for the tabs (ask sanjay before altering this) ---><br />
<ul class="tabs"><br />
<li><a href="#view1">Stage 1</a></li><br />
<li><a href="#view2">Stage 2</a></li><br />
<li><a href="#view3">Stage 3</a></li><br />
<li><a href="#view4">Stage 4</a></li><br />
<li><a href="#view5">Stage 5</a></li><br />
<li><a href="#view6">Stage 6</a></li><br />
<li><a href="#view7">Stage 7</a></li><br />
<li><a href="#view8">Stage 8</a></li><br />
</ul><br />
<div class="tabcontents"><br />
<br />
<!--- This is the overview section ---><br />
<div id="view1"><div class="textTitle"><h4>Stage 01: Extraction of Useful BioBrick Plasmids from iGEM 2014 Distribution Kit</h4></div><br><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<div><strong>Protocols&nbsp;&nbsp;</strong><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">competent cells</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">transformation</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">miniprep</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">digest</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">gel</span></a></div><br />
<p>We began our project by identifying a range of BioBrick parts present in the iGEM 2014 distribution kit which we required as part of our cloning strategy. These parts primarily consisted of both constituitive and inducible promoter systems with ribosome binding sites which we could then use in conjunction with our azo-reductase BioBricks to assemble a functional azo dye degrading gene. We also decided that we would use the Red Florescent Protein expressing BioBrick as a control for any further transformation experiments. As the level of DNA present within each plate of the distribution kit is insufficient to perform digest and ligation reactions on it was necessary to transform each of these plasmids into our NEB5alpha competent cells. After growing our transformed cells overnight we then mini-prepped each of them to obtain BioBrick plasmids at suitable concentrations for future experiments.</p><br><br />
<font size="2"><br />
<table border="1px" width="100%" height="auto"><br />
<thead><br />
<tr><br />
<th> </th><br />
<th> Registry ID </th><br />
<th> Name / Function </th><br />
<th> Antibiotic Resistance </th><br />
<th> Source </th><br />
<th> Size </th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<td> <center>U</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K314103">BBa_K314103</a> </td><br />
<td> &nbsp;IPTG-inducible LacI Expression Cassette </td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 1, Well 4D.</td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K314103">1638 bp</a> </td><br />
</tr><br />
<tr><br />
<td> <center>T</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a> </td><br />
<td> &nbsp;RFP Coding Device </td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 4, Well 4B. </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_J04450">1069 bp</a> </td><br />
</tr><br />
<tr><br />
<td> <center>T</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_R0010">BBa_R0010</a> </td><br />
<td> &nbsp;IPTG-inducible LacI Promoter</td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 3, Well 4G.</td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_R0010">200 bp</a> </td><br />
</tr><br />
<tr><br />
<td> <center>T</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> </td><br />
<td> &nbsp;Ribosomal Binding Site (RBS)</td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 4, Well 1N.</td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_B0034">12 bp</a> </td><br />
</tr><br />
<tr><br />
<td> <center>T</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K518012">BBa_K518012</a> </td><br />
<td> &nbsp;RBS + RFP + Double Terminator</td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 1, Well 18C.</td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K518012">828 bp</a> </td><br />
</tr><br />
<tr><br />
<td> <center>N</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K206000">BBa_K206000</a> </td><br />
<td> &nbsp;pBAD Strong Promoter</td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 3, Well 14A.</td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K206000">130 bp</a> </td><br />
</tr><br />
<tr><br />
<td> <center>! N</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_R0011">BBa_R0011</a> </td><br />
<td> &nbsp;LacI-Regulated, Lambda pL Hybrid Promoter</td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 2, Well 6D.</td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_R0011">55 bp</a> </td><br />
</tr><br />
<tr><br />
<td> <center>! N</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_B0012">BBa_B0012</a> </td><br />
<td> &nbsp;Transcription Terminator for E. coli RNA Polymerase</td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 2, Well 2B.</td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_B0012">41 bp</a> </td><br />
</tr><br />
</tbody><br />
</table><br />
</font><br />
<div><font size="1">Note: U = Used in experiments; T = Used for testing purposes but not for making BioBrick Devices; N = Transformed from Distribution Kits, but not used in experiments; ! = Problematic parts (see Parts Registry), were not used.</font></div><br />
<br />
</div><br />
<br />
<!--- This is the first biobrick ---><br />
<div id="view2"><div class="textTitle"><h4>Stage 02: Identification of Useful Genes for Making New BioBricks</h4></div><br><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<strong>Identifying Azo-Dye Degrading Enzymes</strong><br />
<p>Searching through the literature, we identified a number of bacterial species (including <em>Bacillus subtilis</em> and <em>Pseudomonas sp.</em>) that have proven to degrade azo dye compounds <a href="http://www.ncbi.nlm.nih.gov/pubmed/21655981">[1]</a><a href="http://www.ncbi.nlm.nih.gov/pubmed/24475252">[2]</a><a href="http://www.itqb.unl.pt/martins/index_files/JBC2002.pdf">[3]</a><a href="http://www.ncbi.nlm.nih.gov/pubmed/23820555">[4]</a>. <br><br />
We contacted the <a href="http://www.itqb.unl.pt/martins">Microbial & Enzyme Technology Lab</a> led by Dr Lígia O. Martins at the Universidade Nova de Lisboa, who are currently researching how azo dye degrading enzymes function, and they were keen to collaborate with us on our project. They agreed to send us a set of five plasmids, each containing different genes encoding azo dye degrading enzymes from both <em>B. subtilis</em> and <em>P. putida</em> (including mutated forms found to exhibit enhanced degradation activity), for us to use in our investigations (see Table below). <br><br />
</p><br><br />
<font size="2"><br />
<table border="1px" width="100%" height="auto"><br />
<thead><br />
<tr><br />
<th> Gene ID</th><br />
<th> Name / Function </th><br />
<th> Source </th><br />
<th> Size </th><br />
<th> Plasmid </th><br />
</tr><br />
</thead><br />
<tbody><br />
<!--Lisbon plasmids--><br />
<tr><br />
<td> &nbsp;<a href="http://www.ncbi.nlm.nih.gov/pubmed/21655981">pAzoR</a> </td><br />
<td> &nbsp;FMN-dependent NADH-azoreductase 1 </td><br />
<td> &nbsp;<em>Pseudomonas putida</em> </td><br />
<td> &nbsp;612 bp </td><br />
<td> &nbsp;In expression vector: pET-21a (+) (ampicillin resistant) <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[2] <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[3] </a>, <br>initially cloned between <em>NdeI</em> and <em>BamHI</em> restriction sites. </td><br />
</tr><br />
<tr><br />
<td> &nbsp;<a href="http://www.ncbi.nlm.nih.gov/pubmed/24475252">p1B6</a> </td><br />
<td> &nbsp;AzoR Heat-stable Mutant</td><br />
<td> &nbsp;<em>Pseudomonas putida</em> </td><br />
<td> &nbsp;612 bp </td><br />
<td> &nbsp;In expression vector: pET-21a (+) (ampicillin resistant) <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[2] <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[3] </a>, <br>initially cloned between <em>NdeI</em> and <em>BamHI</em> restriction sites. </td><br />
</tr><br />
<tr><br />
<td> &nbsp;<a href="http://www.itqb.unl.pt/martins/index_files/JBC2002.pdf">pCotA</a> </td><br />
<td> &nbsp;Spore Coat Protein Laccase</td><br />
<td> &nbsp;<em>Bacillus subtilis</em> </td><br />
<td> &nbsp;1542 bp </td><br />
<td> &nbsp;In expression vector: pET-21a (+) (ampicillin resistant (ampR)) <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[2] <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[3] </a>, <br>initially cloned between <strong><em>NheI</em></strong> and <em>BamHI</em> restriction sites. </td><br />
</tr><br />
<tr><br />
<td> &nbsp;<a href="http://www.ncbi.nlm.nih.gov/pubmed/23820555">pBsDyP</a> </td><br />
<td> &nbsp;Dye Decolourising Peroxidase BSU38260</td><br />
<td> &nbsp;<em>Bacillus subtilis</em> </td><br />
<td> &nbsp;1251 bp </td><br />
<td> &nbsp;In expression vector: pET-21a (+) (ampicillin resistant) <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[2] <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[3] </a>, <br>initially cloned between <em>NdeI</em> and <em>BamHI</em> restriction sites. </td><br />
</tr><br />
<tr><br />
<td> &nbsp;<a href="http://www.ncbi.nlm.nih.gov/pubmed/23820555">pPpDyP</a> </td><br />
<td> &nbsp;Dye Decolourising Peroxidase PP_3248 </td><br />
<td> &nbsp;<em>Pseudomonas putida</em> </td><br />
<td> &nbsp;864 bp </td><br />
<td> &nbsp;In expression vector: pET-21a (+) (ampicillin resistant) <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[2] <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[3] </a>, <br>initially cloned between <em>NdeI</em> and <em>BamHI</em> restriction sites. </td><br />
</tr><br />
</tbody><br />
</table><br />
</font><br />
<br><br />
<br />
<a data-tip="true" class="top large" data-tip-content="Here's the gel visualisation showing the B. subtilis genomic DNA!" href="javascript:void(0)" style="width: 20%;float: right;margin-left:1%"><img src="https://static.igem.org/mediawiki/2014/b/b3/UCL_Bsub_Genomic_Extraction.jpeg" style="max-width: 100%;"></a><br />
<strong>Extraction of <em>B. subtilis</em> genomic DNA</strong><br />
<div><strong>Protocols&nbsp;&nbsp;</strong><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">DNA extraction</span></a></div><br />
<p>In the meantime, Helina (in our team), was able to obtain <em>B. subtilis</em> and <em>P. aeruginosa</em> strains for us to test whether we could retrieve azo dye degrading enzymes from their genomes, specifically, the azo-reductase gene (AzoR). This would be the first step for our first azoreductase BioBrick. <br><br />
We extracted the genomic DNA from <em>B. subtilis</em> strain using a Promega Wizard Genomic DNA extraction kit so that we could subsequently amplify the azoreducatase gene (AzoR1) and create our first azoreductase BioBrick. After completing the genomic DNA extracton we ran a gel to show that we had successfully extracted the <i>B. subtilis</i> genomic DNA.</p><br />
<br><br><br />
<br />
<!-- <div class="accordion"><br />
<h4><div class="byline"><i class="icon-user"></i><strong>Extraction of Bacillus Subtilis Genomic DNA</strong></div></h4><br />
<div><br />
<div><strong>Protocols&nbsp;&nbsp;</strong><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">DNA extraction</span></a></div><br />
<p>Our literature search identified a number of bacterial species that have been proven to degrade azo dye compounds including <i>B. subtilis</i> and <i>P. aeruginosa</i>. We were able to obtain a <i>B. subtilis</i> strain for use in our project from ?. We extracted the genomic DNA from this strain using a Promega Wizard Genomic DNA extraction kit so that we could subsequently amplify the azo-reducatase gene (AzoR1) and create our first azo-reductase BioBrick. After completing the genomic DNA extracton we ran a gel to show that we had successfully extracted the <i>B. subtilis</i> genomic DNA.</p><br />
</div><br />
<h4><div class="byline"><i class="icon-user"></i> Adam Denyer, Tanel Ozdemir &nbsp;&nbsp; <i class="icon-time"></i> <abbr class="published" title="June 13, 2014">June 13, 2014</abbr></div></h4><br />
<div><br />
<p>...</p><br />
</div><br />
</div> --><br />
<br />
</div><br />
<br />
<!--- This is the second biobrick ---><br />
<div id="view3"><div class="textTitle"><h4>Stage 03: Transforming E. coli with Azo-Dye Degrading Plasmids from Lisbon</h4></div><br><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<strong>Transforming <em>E. coli</em> with Azo-Dye Degrading Plasmids</strong><br />
<div><strong>Protocols&nbsp;&nbsp;</strong><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">competent cells</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">transformation</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">miniprep</span></a></div><br />
<p>The five azo dye degrading enzymes from Lisbon arrived as the respective genes in pET-21a (+) ampicillin resistant (ampR) expression vectors/plasmids (size: 5443 bp)<a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[1]</a><a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[2]</a>. The DNA concentrations of these plasmids, however, were insufficient to perform PCR amplification, therefore we transformed each into our own <em>E. coli</em> competent cells (grown from NEB DH5&alpha; derivatives). After growing the cells overnight, we made bacterial glycerol stocks and miniprepped the cells to obtain plasmids at sufficient concentrations for further work.</p> <br />
<br><br />
<a data-tip="true" class="top large" data-tip-content="Here's the gel visualisation showing an analytical digest of the plasmids we received from Lisbon!" href="javascript:void(0)" style="width: 40%;float: right;margin-left:1%"><img src="https://static.igem.org/mediawiki/2014/0/08/UCL_23-07-2014_Analytical_Digest_Visualisation.pptx.png" style="max-width: 100%;"></a><br />
<strong>Diagnostic Digest of Azo-Dye Degrading Plasmids</strong><br />
<div><strong>Protocols&nbsp;&nbsp;</strong><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">digest</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">gel</span></a></div><br />
<p>A diagnostic digest was performed to ascertain that these pET-21a (+) plasmids contained the gene we expected. As each plasmid possessed <em>EcoRI</em> and <em>XbaI</em> restriction sites close to the genes of interest, we performed double-digests using these recognition enzymes and predicted the digest fragments. The digestion products were visualised using gel electrophoresis (see image right). </p> <br />
<br><br><br><br><br><br><br />
<br />
</div><br />
<br />
<!--- This is the third biobrick ---><br />
<div id="view4"><div class="textTitle"><h4>Stage 04: Creation of Azo-Reductase BioBrick Parts from Plasmids</h4></div><br><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<div><strong>Protocols&nbsp;&nbsp;</strong><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">digest</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">gel</span></a></div><br />
<p>After isolating our genes of interest we attempted to use PCR as a method of prefix and suffix generation to fit the BioBrick standard assembly.</p><br />
<br/><br />
<p>Achieving a successful PCR proved difficult, this may have been due to poor reagent quality. We repeated the PCR using Taq, phusion and Pfu polymerases. We took an alternative route successfully used directionless ligation to generate the prefix and suffix for 1B6. Taq polymerase eventually gave us a successful generation of prefix and suffix for BsDyP, AzoR and ispB. Unfortunately, due to time constraints we were unable to implement site directed mutagenesis on 1B6 required to remove two illegal PstI and therefore did not submitt the part to the registry.</p><br />
<br/><br />
<br />
<strong>Diagnostic Digest of Azo-Dye Degrading Plasmids</strong><br />
<div><strong>Protocols&nbsp;&nbsp;</strong></div><br />
<p>We confirmed the success of the PCR through gel visualisation, comparing PCR products with and without prefix and suffix.</p><br />
</div><br />
<br />
<!--- This is the fourth biobrick ---><br />
<div id="view5"><div class="textTitle"><h4>Stage 05: Creation of Azo-Reductase BioBrick Parts from Plasmids</h4></div><br><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>We decided to assemble both BsDyP and ispB in a LacI cassette, inducible by IPTG. <br />
Issues with inconclusive antibiotic effectivity led to major delays in construction of these composite parts. We first had to prove our antibiotics were functioning properly before making progress on our project.</p><br/><br />
<p>We confirmed the construction of our BsDyP and ispB cassettes using analytical gel digest cutting at sites E and P.</p><br />
</div><br />
<br />
<!--- This is the fifth biobrick ---><br />
<div id="view6"><div class="textTitle"><h4>Stage 06: Diagnostic Digest of Azo-Reductase BioBrick Parts</h4></div><br><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>...</p><br><br />
</div><br />
<br />
<!--- This is the fifth biobrick ---><br />
<div id="view7"><div class="textTitle"><h4>Stage 07: Assembling Azo-Reductase BioBrick Device(s)</h4></div><br><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>...</p><br><br />
<font size="2"><br />
<table border="1px" width="100%" height="auto"><br />
<thead><br />
<tr><br />
<th> </th><br />
<th> Registry ID </th><br />
<th> Gene ID</th><br />
<th> Name / Function </th><br />
<th> Source </th><br />
<th> Size </th><br />
<th> Status </th><br />
</tr><br />
</thead><br />
<tbody><br />
<!--Lisbon plasmids--><br />
<tr><br />
<td> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336000">BBa_K1336000</a> </td><br />
<td> &nbsp;AzoR </td><br />
<td> &nbsp;FMN-dependent NADH-azoreductase 1 </td><br />
<td> &nbsp;<em>Pseudomonas putida</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336000">612 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Primers">In Progress</a>]: primers designed </td><br />
</tr><br />
<tr><br />
<td> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336001">BBa_K1336001</a> </td><br />
<td> &nbsp;1B6 </td><br />
<td> &nbsp;AzoR heat-stable mutant</td><br />
<td> &nbsp;<em>Pseudomonas putida</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336001">612 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Experiments">In Progress</a>]: to remove 2 illegal PstI sites </td><br />
</tr><br />
<tr><br />
<td> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336002">BBa_K1336002</a> </td><br />
<td> &nbsp;CotA </td><br />
<td> &nbsp;Spore Coat Protein Laccase</td><br />
<td> &nbsp;<em>Bacillus subtilis</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336002">1542 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Primers">In Progress</a>]: primers designed </td><br />
</tr><br />
<tr><br />
<td> <center><img src="https://static.igem.org/mediawiki/2014/e/e0/UCL_Bronze-metal-star.jpg" width="25px"></center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336003">BBa_K1336003</a> </td><br />
<td> &nbsp;BsDyP </td><br />
<td> &nbsp;Dye Decolourising Peroxidase BSU38260</td><br />
<td> &nbsp;<em>Bacillus subtilis</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336003">1251 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Experiments">New BioBrick Part</a>]: submitted </td><br />
</tr><br />
<tr><br />
<td> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336004">BBa_K1336004</a> </td><br />
<td> &nbsp;PpDyP </td><br />
<td> &nbsp;Dye Decolourising Peroxidase PP_3248 </td><br />
<td> &nbsp;<em>Pseudomonas putida</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336004">864 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Primers">In Progress</a>]: primers designed </td><br />
</tr><br />
<tr><br />
<td> <center><img src="https://static.igem.org/mediawiki/2014/e/e0/UCL_Bronze-metal-star.jpg" width="25px"></center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336005">BBa_K1336005</a> </td><br />
<td> &nbsp;ispB RNAi </td><br />
<td> &nbsp;RNAi of Octaprenyl Diphosphate <br>Synthase fragment </td><br />
<td> &nbsp;<em>Escherichia coli, K12 strain</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336005">562 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Experiment">New BioBrick Part</a>]: submitted </td><br />
</tr><br />
<tr><br />
<td> <center><img src="https://static.igem.org/mediawiki/2014/e/e0/UCL_Bronze-metal-star.jpg" width="25px"></center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336006">BBa_K1336006</a> </td><br />
<td> &nbsp;LacIEC+ispB </td><br />
<td> &nbsp;IPTG inducible ispB RNAi </td><br />
<td> &nbsp;<em>Escherichia coli, K12 strain </em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336006">2208 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Experiment">New BioBrick Device</a>]: submitted </td><br />
</tr><br />
<tr><br />
<td> <center><img src="https://static.igem.org/mediawiki/2014/e/e0/UCL_Bronze-metal-star.jpg" width="25px"></center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336007">BBa_K1336007</a> </td><br />
<td> &nbsp;LacIEC+BsDyP </td><br />
<td> &nbsp;IPTG inducible BsDyP </td><br />
<td> &nbsp;<em>Bacillus subtilis</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336007">2895 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Experiment">New BioBrick Device</a>]: submitted </td><br />
</tr><br />
<tr><br />
<td> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K729006">BBa_K729006</a> </td><br />
<td> &nbsp;CueO </td><br />
<td> &nbsp;Laccase </td><br />
<td> &nbsp;<em>Escherichia coli </em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K729006">1612 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Experiment">In Progress</a>]: ascertaining identity </td><br />
</tr><br />
<tr><br />
<td> <center>(<img src="https://static.igem.org/mediawiki/2014/3/36/UCL_Gold-metal-star.jpg" width="25px">)</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K500000">BBa_K500000</a> </td><br />
<td> &nbsp;LiP </td><br />
<td> &nbsp;Lignin Peroxidase </td><br />
<td> &nbsp;<em>Phanerochaete chrysosporium</em> </td> <!-- <br>(White-Rot Fungi) --><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K500000">1116 bp</a> </td> <!--Check size!--><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Results">Improved Characterisation</a>]: toxicity issues in gene synthesis. <br>&nbsp;[<a href="/Team:UCL/Science/Experiment">In Progress</a>]: to subclone into pSB1C3/pSB3C5. </td><br />
</tr><br />
<tr><br />
<td> <center><img src="https://static.igem.org/mediawiki/2014/3/36/UCL_Gold-metal-star.jpg" width="25px"></center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K729004">BBa_K729004</a> </td><br />
<td> &nbsp;nucB </td><br />
<td> &nbsp;Extracellular nuclease </td><br />
<td> &nbsp;<em>Staphylococcus aureus</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K729004">561 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Results">Improved Function</a>] </td><br />
</tr><br />
</tbody><br />
</table><br />
</font><br />
</div><br />
<br />
<!--- This is the fifth biobrick ---><br />
<div id="view8"><div class="textTitle"><h4>Stage 08: Characterisation of Azo-Reductase BioBrick Devices</h4></div><br><br />
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<p>...</p><br><br />
<div><br />
<h4><a name="Expt">Placeholder. Will be removed.</a></h4><br />
<strong>Protocols&nbsp;&nbsp;</strong><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">PCR</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">analytical digest</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">gel</span></a><br />
(<a href="/Team:UCL/Science/Proto"><span class="label label-warning">digest</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">ligation</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">competent cells</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">transformation</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">miniprep</span></a>)<br />
<br><br />
<p>[Insert table of Our Genes]</p><br />
<br><br />
<div class="accordion"><br />
<h4><div class="byline"><i class="icon-user"></i> Adam Denyer, Tanel Ozdemir &nbsp;&nbsp; <i class="icon-time"></i> <abbr class="published" title="June 13, 2014">June 13, 2014</abbr></div></h4><br />
<div><br />
<p>...</p><br />
</div><br />
<h4><div class="byline"><i class="icon-user"></i> Adam Denyer, Tanel Ozdemir &nbsp;&nbsp; <i class="icon-time"></i> <abbr class="published" title="June 13, 2014">June 13, 2014</abbr></div></h4><br />
<div><br />
<p>...</p><br />
</div><br />
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{{:Team:UCL/Template:footerx}}</div>Ningluhttp://2014.igem.org/Team:UCL/Science/ExperimentTeam:UCL/Science/Experiment2014-10-18T01:18:59Z<p>Ninglu: </p>
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<li><a href="#view1">Stage 1</a></li><br />
<li><a href="#view2">Stage 2</a></li><br />
<li><a href="#view3">Stage 3</a></li><br />
<li><a href="#view4">Stage 4</a></li><br />
<li><a href="#view5">Stage 5</a></li><br />
<li><a href="#view6">Stage 6</a></li><br />
<li><a href="#view7">Stage 7</a></li><br />
<li><a href="#view8">Stage 8</a></li><br />
</ul><br />
<div class="tabcontents"><br />
<br />
<!--- This is the overview section ---><br />
<div id="view1"><div class="textTitle"><h4>Stage 01: Extraction of Useful BioBrick Plasmids from iGEM 2014 Distribution Kit</h4></div><br><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<div><strong>Protocols&nbsp;&nbsp;</strong><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">competent cells</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">transformation</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">miniprep</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">digest</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">gel</span></a></div><br />
<p>We began our project by identifying a range of BioBrick parts present in the iGEM 2014 distribution kit which we required as part of our cloning strategy. These parts primarily consisted of both constituitive and inducible promoter systems with ribosome binding sites which we could then use in conjunction with our azo-reductase BioBricks to assemble a functional azo dye degrading gene. We also decided that we would use the Red Florescent Protein expressing BioBrick as a control for any further transformation experiments. As the level of DNA present within each plate of the distribution kit is insufficient to perform digest and ligation reactions on it was necessary to transform each of these plasmids into our NEB5alpha competent cells. After growing our transformed cells overnight we then mini-prepped each of them to obtain BioBrick plasmids at suitable concentrations for future experiments.</p><br><br />
<font size="2"><br />
<table border="1px" width="100%" height="auto"><br />
<thead><br />
<tr><br />
<th> </th><br />
<th> Registry ID </th><br />
<th> Name / Function </th><br />
<th> Antibiotic Resistance </th><br />
<th> Source </th><br />
<th> Size </th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<td> <center>U</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K314103">BBa_K314103</a> </td><br />
<td> &nbsp;IPTG-inducible LacI Expression Cassette </td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 1, Well 4D.</td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K314103">1638 bp</a> </td><br />
</tr><br />
<tr><br />
<td> <center>T</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a> </td><br />
<td> &nbsp;RFP Coding Device </td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 4, Well 4B. </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_J04450">1069 bp</a> </td><br />
</tr><br />
<tr><br />
<td> <center>T</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_R0010">BBa_R0010</a> </td><br />
<td> &nbsp;IPTG-inducible LacI Promoter</td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 3, Well 4G.</td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_R0010">200 bp</a> </td><br />
</tr><br />
<tr><br />
<td> <center>T</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> </td><br />
<td> &nbsp;Ribosomal Binding Site (RBS)</td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 4, Well 1N.</td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_B0034">12 bp</a> </td><br />
</tr><br />
<tr><br />
<td> <center>T</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K518012">BBa_K518012</a> </td><br />
<td> &nbsp;RBS + RFP + Double Terminator</td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 1, Well 18C.</td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K518012">828 bp</a> </td><br />
</tr><br />
<tr><br />
<td> <center>N</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K206000">BBa_K206000</a> </td><br />
<td> &nbsp;pBAD Strong Promoter</td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 3, Well 14A.</td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K206000">130 bp</a> </td><br />
</tr><br />
<tr><br />
<td> <center>! N</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_R0011">BBa_R0011</a> </td><br />
<td> &nbsp;LacI-Regulated, Lambda pL Hybrid Promoter</td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 2, Well 6D.</td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_R0011">55 bp</a> </td><br />
</tr><br />
<tr><br />
<td> <center>! N</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_B0012">BBa_B0012</a> </td><br />
<td> &nbsp;Transcription Terminator for E. coli RNA Polymerase</td><br />
<td> &nbsp;Chloramphenicol</td><br />
<td> &nbsp;Spring 2014 BioBrick Distribution. Plate 2, Well 2B.</td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_B0012">41 bp</a> </td><br />
</tr><br />
</tbody><br />
</table><br />
</font><br />
<div><font size="1">Note: U = Used in experiments; T = Used for testing purposes but not for making BioBrick Devices; N = Transformed from Distribution Kits, but not used in experiments; ! = Problematic parts (see Parts Registry), were not used.</font></div><br />
<br />
</div><br />
<br />
<!--- This is the first biobrick ---><br />
<div id="view2"><div class="textTitle"><h4>Stage 02: Identification of Useful Genes for Making New BioBricks</h4></div><br><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<strong>Identifying Azo-Dye Degrading Enzymes</strong><br />
<p>Searching through the literature, we identified a number of bacterial species (including <em>Bacillus subtilis</em> and <em>Pseudomonas sp.</em>) that have proven to degrade azo dye compounds <a href="http://www.ncbi.nlm.nih.gov/pubmed/21655981">[1]</a><a href="http://www.ncbi.nlm.nih.gov/pubmed/24475252">[2]</a><a href="http://www.itqb.unl.pt/martins/index_files/JBC2002.pdf">[3]</a><a href="http://www.ncbi.nlm.nih.gov/pubmed/23820555">[4]</a>. <br><br />
We contacted the <a href="http://www.itqb.unl.pt/martins">Microbial & Enzyme Technology Lab</a> led by Dr Lígia O. Martins at the Universidade Nova de Lisboa, who are currently researching how azo dye degrading enzymes function, and they were keen to collaborate with us on our project. They agreed to send us a set of five plasmids, each containing different genes encoding azo dye degrading enzymes from both <em>B. subtilis</em> and <em>P. putida</em> (including mutated forms found to exhibit enhanced degradation activity), for us to use in our investigations (see Table below). <br><br />
</p><br><br />
<font size="2"><br />
<table border="1px" width="100%" height="auto"><br />
<thead><br />
<tr><br />
<th> Gene ID</th><br />
<th> Name / Function </th><br />
<th> Source </th><br />
<th> Size </th><br />
<th> Plasmid </th><br />
</tr><br />
</thead><br />
<tbody><br />
<!--Lisbon plasmids--><br />
<tr><br />
<td> &nbsp;<a href="http://www.ncbi.nlm.nih.gov/pubmed/21655981">pAzoR</a> </td><br />
<td> &nbsp;FMN-dependent NADH-azoreductase 1 </td><br />
<td> &nbsp;<em>Pseudomonas putida</em> </td><br />
<td> &nbsp;612 bp </td><br />
<td> &nbsp;In expression vector: pET-21a (+) (ampicillin resistant) <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[2] <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[3] </a>, <br>initially cloned between <em>NdeI</em> and <em>BamHI</em> restriction sites. </td><br />
</tr><br />
<tr><br />
<td> &nbsp;<a href="http://www.ncbi.nlm.nih.gov/pubmed/24475252">p1B6</a> </td><br />
<td> &nbsp;AzoR Heat-stable Mutant</td><br />
<td> &nbsp;<em>Pseudomonas putida</em> </td><br />
<td> &nbsp;612 bp </td><br />
<td> &nbsp;In expression vector: pET-21a (+) (ampicillin resistant) <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[2] <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[3] </a>, <br>initially cloned between <em>NdeI</em> and <em>BamHI</em> restriction sites. </td><br />
</tr><br />
<tr><br />
<td> &nbsp;<a href="http://www.itqb.unl.pt/martins/index_files/JBC2002.pdf">pCotA</a> </td><br />
<td> &nbsp;Spore Coat Protein Laccase</td><br />
<td> &nbsp;<em>Bacillus subtilis</em> </td><br />
<td> &nbsp;1542 bp </td><br />
<td> &nbsp;In expression vector: pET-21a (+) (ampicillin resistant (ampR)) <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[2] <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[3] </a>, <br>initially cloned between <strong><em>NheI</em></strong> and <em>BamHI</em> restriction sites. </td><br />
</tr><br />
<tr><br />
<td> &nbsp;<a href="http://www.ncbi.nlm.nih.gov/pubmed/23820555">pBsDyP</a> </td><br />
<td> &nbsp;Dye Decolourising Peroxidase BSU38260</td><br />
<td> &nbsp;<em>Bacillus subtilis</em> </td><br />
<td> &nbsp;1251 bp </td><br />
<td> &nbsp;In expression vector: pET-21a (+) (ampicillin resistant) <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[2] <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[3] </a>, <br>initially cloned between <em>NdeI</em> and <em>BamHI</em> restriction sites. </td><br />
</tr><br />
<tr><br />
<td> &nbsp;<a href="http://www.ncbi.nlm.nih.gov/pubmed/23820555">pPpDyP</a> </td><br />
<td> &nbsp;Dye Decolourising Peroxidase PP_3248 </td><br />
<td> &nbsp;<em>Pseudomonas putida</em> </td><br />
<td> &nbsp;864 bp </td><br />
<td> &nbsp;In expression vector: pET-21a (+) (ampicillin resistant) <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[2] <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[3] </a>, <br>initially cloned between <em>NdeI</em> and <em>BamHI</em> restriction sites. </td><br />
</tr><br />
</tbody><br />
</table><br />
</font><br />
<br><br />
<br />
<a data-tip="true" class="top large" data-tip-content="Here's the gel visualisation showing the B. subtilis genomic DNA!" href="javascript:void(0)" style="width: 20%;float: right;margin-left:1%"><img src="https://static.igem.org/mediawiki/2014/b/b3/UCL_Bsub_Genomic_Extraction.jpeg" style="max-width: 100%;"></a><br />
<strong>Extraction of <em>B. subtilis</em> genomic DNA</strong><br />
<div><strong>Protocols&nbsp;&nbsp;</strong><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">DNA extraction</span></a></div><br />
<p>In the meantime, Helina (in our team), was able to obtain <em>B. subtilis</em> and <em>P. aeruginosa</em> strains for us to test whether we could retrieve azo dye degrading enzymes from their genomes, specifically, the azo-reductase gene (AzoR). This would be the first step for our first azoreductase BioBrick. <br><br />
We extracted the genomic DNA from <em>B. subtilis</em> strain using a Promega Wizard Genomic DNA extraction kit so that we could subsequently amplify the azoreducatase gene (AzoR1) and create our first azoreductase BioBrick. After completing the genomic DNA extracton we ran a gel to show that we had successfully extracted the <i>B. subtilis</i> genomic DNA.</p><br />
<br><br><br />
<br />
<!-- <div class="accordion"><br />
<h4><div class="byline"><i class="icon-user"></i><strong>Extraction of Bacillus subtilis genomic DNA</strong></div></h4><br />
<div><br />
<div><strong>Protocols&nbsp;&nbsp;</strong><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">DNA extraction</span></a></div><br />
<p>Our literature search identified a number of bacterial species that have been proven to degrade azo dye compounds including <i>B. subtilis</i> and <i>P. aeruginosa</i>. We were able to obtain a <i>B. subtilis</i> strain for use in our project from ?. We extracted the genomic DNA from this strain using a Promega Wizard Genomic DNA extraction kit so that we could subsequently amplify the azo-reducatase gene (AzoR1) and create our first azo-reductase BioBrick. After completing the genomic DNA extracton we ran a gel to show that we had successfully extracted the <i>B. subtilis</i> genomic DNA.</p><br />
</div><br />
<h4><div class="byline"><i class="icon-user"></i> Adam Denyer, Tanel Ozdemir &nbsp;&nbsp; <i class="icon-time"></i> <abbr class="published" title="June 13, 2014">June 13, 2014</abbr></div></h4><br />
<div><br />
<p>...</p><br />
</div><br />
</div> --><br />
<br />
</div><br />
<br />
<!--- This is the second biobrick ---><br />
<div id="view3"><div class="textTitle"><h4>Stage 03: Transforming E. coli with Azo-Dye Degrading Plasmids from Lisbon</h4></div><br><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<strong>Transforming <em>E. coli</em> with Azo-Dye Degrading Plasmids</strong><br />
<div><strong>Protocols&nbsp;&nbsp;</strong><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">competent cells</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">transformation</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">miniprep</span></a></div><br />
<p>The five azo dye degrading enzymes from Lisbon arrived as the respective genes in pET-21a (+) ampicillin resistant (ampR) expression vectors/plasmids (size: 5443 bp)<a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[1]</a><a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[2]</a>. The DNA concentrations of these plasmids, however, were insufficient to perform PCR amplification, therefore we transformed each into our own <em>E. coli</em> competent cells (grown from NEB DH5&alpha; derivatives). After growing the cells overnight, we made bacterial glycerol stocks and miniprepped the cells to obtain plasmids at sufficient concentrations for further work.</p> <br />
<br><br />
<a data-tip="true" class="top large" data-tip-content="Here's the gel visualisation showing an analytical digest of the plasmids we received from Lisbon!" href="javascript:void(0)" style="width: 40%;float: right;margin-left:1%"><img src="https://static.igem.org/mediawiki/2014/0/08/UCL_23-07-2014_Analytical_Digest_Visualisation.pptx.png" style="max-width: 100%;"></a><br />
<strong>Diagnostic Digest of Azo-Dye Degrading Plasmids</strong><br />
<div><strong>Protocols&nbsp;&nbsp;</strong><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">digest</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">gel</span></a></div><br />
<p>A diagnostic digest was performed to ascertain that these pET-21a (+) plasmids contained the gene we expected. As each plasmid possessed <em>EcoRI</em> and <em>XbaI</em> restriction sites close to the genes of interest, we performed double-digests using these recognition enzymes and predicted the digest fragments. The digestion products were visualised using gel electrophoresis (see image right). </p> <br />
<br><br><br><br><br><br><br />
<br />
</div><br />
<br />
<!--- This is the third biobrick ---><br />
<div id="view4"><div class="textTitle"><h4>Stage 04: Creation of Azo-Reductase BioBrick Parts from Plasmids</h4></div><br><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<div><strong>Protocols&nbsp;&nbsp;</strong><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">digest</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">gel</span></a></div><br />
<p>After isolating our genes of interest we attempted to use PCR as a method of prefix and suffix generation to fit the BioBrick standard assembly.</p><br />
<br/><br />
<p>Achieving a successful PCR proved difficult, this may have been due to poor reagent quality. We repeated the PCR using Taq, phusion and Pfu polymerases. We took an alternative route successfully used directionless ligation to generate the prefix and suffix for 1B6. Taq polymerase eventually gave us a successful generation of prefix and suffix for BsDyP, AzoR and ispB. Unfortunately, due to time constraints we were unable to implement site directed mutagenesis on 1B6 required to remove two illegal PstI and therefore did not submitt the part to the registry.</p><br />
<br/><br />
<br />
<strong>Diagnostic Digest of Azo-Dye Degrading Plasmids</strong><br />
<div><strong>Protocols&nbsp;&nbsp;</strong></div><br />
<p>We confirmed the success of the PCR through gel visualisation, comparing PCR products with and without prefix and suffix.</p><br />
</div><br />
<br />
<!--- This is the fourth biobrick ---><br />
<div id="view5"><div class="textTitle"><h4>Stage 05: Creation of Azo-Reductase BioBrick Parts from Plasmids</h4></div><br><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>We decided to assemble both BsDyP and ispB in a LacI cassette, inducible by IPTG. <br />
Issues with inconclusive antibiotic effectivity led to major delays in construction of these composite parts. We first had to prove our antibiotics were functioning properly before making progress on our project.</p><br/><br />
<p>We confirmed the construction of our BsDyP and ispB cassettes using analytical gel digest cutting at sites E and P.</p><br />
</div><br />
<br />
<!--- This is the fifth biobrick ---><br />
<div id="view6"><div class="textTitle"><h4>Stage 06: Diagnostic Digest of Azo-Reductase BioBrick Parts</h4></div><br><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>...</p><br><br />
</div><br />
<br />
<!--- This is the fifth biobrick ---><br />
<div id="view7"><div class="textTitle"><h4>Stage 07: Assembling Azo-Reductase BioBrick Device(s)</h4></div><br><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>...</p><br><br />
<font size="2"><br />
<table border="1px" width="100%" height="auto"><br />
<thead><br />
<tr><br />
<th> </th><br />
<th> Registry ID </th><br />
<th> Gene ID</th><br />
<th> Name / Function </th><br />
<th> Source </th><br />
<th> Size </th><br />
<th> Status </th><br />
</tr><br />
</thead><br />
<tbody><br />
<!--Lisbon plasmids--><br />
<tr><br />
<td> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336000">BBa_K1336000</a> </td><br />
<td> &nbsp;AzoR </td><br />
<td> &nbsp;FMN-dependent NADH-azoreductase 1 </td><br />
<td> &nbsp;<em>Pseudomonas putida</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336000">612 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Primers">In Progress</a>]: primers designed </td><br />
</tr><br />
<tr><br />
<td> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336001">BBa_K1336001</a> </td><br />
<td> &nbsp;1B6 </td><br />
<td> &nbsp;AzoR heat-stable mutant</td><br />
<td> &nbsp;<em>Pseudomonas putida</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336001">612 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Experiments">In Progress</a>]: to remove 2 illegal PstI sites </td><br />
</tr><br />
<tr><br />
<td> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336002">BBa_K1336002</a> </td><br />
<td> &nbsp;CotA </td><br />
<td> &nbsp;Spore Coat Protein Laccase</td><br />
<td> &nbsp;<em>Bacillus subtilis</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336002">1542 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Primers">In Progress</a>]: primers designed </td><br />
</tr><br />
<tr><br />
<td> <center><img src="https://static.igem.org/mediawiki/2014/e/e0/UCL_Bronze-metal-star.jpg" width="25px"></center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336003">BBa_K1336003</a> </td><br />
<td> &nbsp;BsDyP </td><br />
<td> &nbsp;Dye Decolourising Peroxidase BSU38260</td><br />
<td> &nbsp;<em>Bacillus subtilis</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336003">1251 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Experiments">New BioBrick Part</a>]: submitted </td><br />
</tr><br />
<tr><br />
<td> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336004">BBa_K1336004</a> </td><br />
<td> &nbsp;PpDyP </td><br />
<td> &nbsp;Dye Decolourising Peroxidase PP_3248 </td><br />
<td> &nbsp;<em>Pseudomonas putida</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336004">864 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Primers">In Progress</a>]: primers designed </td><br />
</tr><br />
<tr><br />
<td> <center><img src="https://static.igem.org/mediawiki/2014/e/e0/UCL_Bronze-metal-star.jpg" width="25px"></center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336005">BBa_K1336005</a> </td><br />
<td> &nbsp;ispB RNAi </td><br />
<td> &nbsp;RNAi of Octaprenyl Diphosphate <br>Synthase fragment </td><br />
<td> &nbsp;<em>Escherichia coli, K12 strain</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336005">562 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Experiment">New BioBrick Part</a>]: submitted </td><br />
</tr><br />
<tr><br />
<td> <center><img src="https://static.igem.org/mediawiki/2014/e/e0/UCL_Bronze-metal-star.jpg" width="25px"></center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336006">BBa_K1336006</a> </td><br />
<td> &nbsp;LacIEC+ispB </td><br />
<td> &nbsp;IPTG inducible ispB RNAi </td><br />
<td> &nbsp;<em>Escherichia coli, K12 strain </em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336006">2208 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Experiment">New BioBrick Device</a>]: submitted </td><br />
</tr><br />
<tr><br />
<td> <center><img src="https://static.igem.org/mediawiki/2014/e/e0/UCL_Bronze-metal-star.jpg" width="25px"></center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K1336007">BBa_K1336007</a> </td><br />
<td> &nbsp;LacIEC+BsDyP </td><br />
<td> &nbsp;IPTG inducible BsDyP </td><br />
<td> &nbsp;<em>Bacillus subtilis</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K1336007">2895 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Experiment">New BioBrick Device</a>]: submitted </td><br />
</tr><br />
<tr><br />
<td> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K729006">BBa_K729006</a> </td><br />
<td> &nbsp;CueO </td><br />
<td> &nbsp;Laccase </td><br />
<td> &nbsp;<em>Escherichia coli </em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K729006">1612 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Experiment">In Progress</a>]: ascertaining identity </td><br />
</tr><br />
<tr><br />
<td> <center>(<img src="https://static.igem.org/mediawiki/2014/3/36/UCL_Gold-metal-star.jpg" width="25px">)</center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K500000">BBa_K500000</a> </td><br />
<td> &nbsp;LiP </td><br />
<td> &nbsp;Lignin Peroxidase </td><br />
<td> &nbsp;<em>Phanerochaete chrysosporium</em> </td> <!-- <br>(White-Rot Fungi) --><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K500000">1116 bp</a> </td> <!--Check size!--><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Results">Improved Characterisation</a>]: toxicity issues in gene synthesis. <br>&nbsp;[<a href="/Team:UCL/Science/Experiment">In Progress</a>]: to subclone into pSB1C3/pSB3C5. </td><br />
</tr><br />
<tr><br />
<td> <center><img src="https://static.igem.org/mediawiki/2014/3/36/UCL_Gold-metal-star.jpg" width="25px"></center> </td><br />
<td> &nbsp;<a href="http://parts.igem.org/Part:BBa_K729004">BBa_K729004</a> </td><br />
<td> &nbsp;nucB </td><br />
<td> &nbsp;Extracellular nuclease </td><br />
<td> &nbsp;<em>Staphylococcus aureus</em> </td><br />
<td> &nbsp;<a href="/Team:UCL/Science/Sequences#BBa_K729004">561 bp</a> </td><br />
<td> &nbsp;[<a href="/Team:UCL/Science/Results">Improved Function</a>] </td><br />
</tr><br />
</tbody><br />
</table><br />
</font><br />
</div><br />
<br />
<!--- This is the fifth biobrick ---><br />
<div id="view8"><div class="textTitle"><h4>Stage 08: Characterisation of Azo-Reductase BioBrick Devices</h4></div><br><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>...</p><br><br />
<div><br />
<h4><a name="Expt">Placeholder. Will be removed.</a></h4><br />
<strong>Protocols&nbsp;&nbsp;</strong><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">PCR</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">analytical digest</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">gel</span></a><br />
(<a href="/Team:UCL/Science/Proto"><span class="label label-warning">digest</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">ligation</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">competent cells</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">transformation</span></a><br />
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">miniprep</span></a>)<br />
<br><br />
<p>[Insert table of Our Genes]</p><br />
<br><br />
<div class="accordion"><br />
<h4><div class="byline"><i class="icon-user"></i> Adam Denyer, Tanel Ozdemir &nbsp;&nbsp; <i class="icon-time"></i> <abbr class="published" title="June 13, 2014">June 13, 2014</abbr></div></h4><br />
<div><br />
<p>...</p><br />
</div><br />
<h4><div class="byline"><i class="icon-user"></i> Adam Denyer, Tanel Ozdemir &nbsp;&nbsp; <i class="icon-time"></i> <abbr class="published" title="June 13, 2014">June 13, 2014</abbr></div></h4><br />
<div><br />
<p>...</p><br />
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{{:Team:UCL/Template:footerx}}</div>Ningluhttp://2014.igem.org/Team:UCL/Project/BiobricksTeam:UCL/Project/Biobricks2014-10-18T00:55:49Z<p>Ninglu: </p>
<hr />
<div>{{:Team:UCL/Template:headerx}}<br />
{{:Team:UCL/Template:BioprocessStyles}}<br />
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<!---<br />
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<div id="BPimagewrapperHeader"><br />
<img src="https://static.igem.org/mediawiki/2014/9/98/UCLBiobricksHeaderOran.jpg" width="100%" height="auto" alt="BioBricks" /><br />
</div><br />
<br />
<div class="textArena"><br />
<!--- This is the coding for the tabs (ask sanjay before altering this) ---><br />
<ul class="tabs"><br />
<li><a href="#view1">UCL iGEM 2014</a></li><br />
<li><a href="#view2">Azoreductase</a></li><br />
<li><a href="#view3">Laccase</a></li><br />
<li><a href="#view4">Lignin Peroxidase</a></li><br />
<li><a href="#view5">Bacterial Peroxidases</a></li><br />
<li><a href="#view6">ispB asRNA</a></li><br />
<li><a href="#view7">Nuclease</a></li><br />
</ul><br />
<div class="tabcontents"><br />
<br />
<!--- This is the overview section ---><br />
<div id="view1"><div class="textTitle"><h4>Our BioBricks & how they lead to azo degradation</h4></div><br><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><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>We have strove to complete a synthetic azo dye decolourising device in E. coli which incorporates several different independent enzymes that act on azo dyes and their breakdown products to create less toxic chemicals. 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.<br />
</p><br />
<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 />
<br><br />
<p> 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.<br />
</p><b>toxicity levels</b></a>.<br />
<br><br><br />
<p> 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.<br />
To ensure that the process is entirely bio-safe we have designed a xeno-biological modules that ensures that beyond the bioreactor our organism could not survive.<br />
</p><br />
</div><br />
<br />
<!--- This is the first biobrick ---><br />
<div id="view2"><div class="textTitle"><h4>Azoreductase (BBa_K1336000)</h4></div><br><br />
<!-- This is the biobrick image --><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." 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><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>This enzyme was isolated from Bacillus subtilis, although it is also found in other bacterial species. It<br />
starts the degradation of azo dyes by reductively cleaving the azo bond.<br />
</p><br />
<p>This azo cleavage, does not only occur with azo dyes, but also with other molecules like Sulfasalazine. We isolated this enzyme from B. subtilis and converted it to BioBrick format via polymerase chain reaction (PCR). However the site directed mutagenesis did not successfully remove the illegal restriction sites and therefore we could not characterise the effects.<br />
</p><br />
<div class="textTitle"><h4>Azoreductase 1B6 (BBa_K1336001)</h4></div><br><br />
<!-- This is the biobrick image --><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." 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><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>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.<br />
</p><br />
<br />
<p>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.<br />
<p> <br />
<br />
<p>AzoR creates two or more aromatic amines (dependent on the number of azo bonds in the molecule) which are carcinogens. This is therefore only the first step of degradation. Some useful aromatic amines will be filtered off and sold as feedstock products for the fragrance industry which is further explained in the bioprocessing pages.<br />
</p> <br />
<br />
<img src="https://static.igem.org/mediawiki/2014/0/0b/SSAzoReductase.png"><br />
<br><br />
</div><br />
<br />
<!--- This is the second biobrick ---><br />
<div id="view3"><div class="textTitle"><h4>Spore Coat Protein Laccase (BBa_K1336002)</h4></div><br><br />
<!-- This is the biobrick image --><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." href="javascript:void(0)" style="width: 30%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/f/f6/UCL_BBa_K1336002.png" style="max-width: 100%;"></a><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>The laccase enzyme is a very non-specific oxidising enzyme. We intend to use it in concert with and as a second step to the azo reductase action to create no toxic products. Laccase will:<br />
<ul><br />
<li><br />
Break down azo bonds in specific azo dyes to non-toxic compounds <br />
</li><br />
<li>Polymerise non-specific azoreductase breakdown products to filterable, non toxic compounds<br />
</li><br />
<li>Oxidise specific azoreductase breakdown products to non-toxic compounds.<br />
</li><br />
</ul><br />
<p>The spanning range of reactions that take place in laccase are largely due to the unspecific copper mediated oxidising active site. Known reaction products are below, however these are known to break down further in ways not yet tested via NMR. <br />
</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/4e/SSlaccase1.png"><br />
<img src="https://static.igem.org/mediawiki/2014/e/eb/SSLaccase2.png"><br />
<p>Despite laccase’s unspecific active site, it cannot break down sulphonated dyes and hence oxidation of those must be left for the peroxidases (see next tab.)<br />
</p><br><br />
</div><br />
<br />
<!--- This is the third biobrick ---><br />
<div id="view4"><div class="textTitle"><h4>Lignin Peroxidase (BBa_K500000)</h4></div><br><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." href="javascript:void(0)" style="width: 30%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/f/f1/UCL_BBa_K500000.png" style="max-width: 100%;"></a><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<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><br />
</div><br />
<br />
<!--- This is the fourth biobrick ---><br />
<div id="view5"><div class="textTitle"><h4><em>Bacillus subtilis</em> dye-decolorizing peroxidase (BsDyP) (BBa_K1336003)</h4></div><br><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." 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><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<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 />
<div class="textTitle"><h4><em>Pseudomonas putida</em> MET94 dye-decolorizing peroxidase (PpDyP) (BBa_K1336004)</h4></div><br><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." 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><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<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><br />
</div><br />
<br />
<!--- This is the fifth biobrick ---><br />
<div id="view6"><div class="textTitle"><h4>Octaprenyl Diphosphate Synthase (ispB) (BBa_K1336005)</h4></div><br><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." 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><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<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><br />
</div><br />
<br />
<!--- This is the fifth biobrick ---><br />
<div id="view7"><div class="textTitle"><h4>Extracellular Nuclease (nucB) (BBa_K729004)</h4></div><br><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." href="javascript:void(0)" style="width: 30%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/e/ec/UCL_BBa_K729004.png" style="max-width: 100%;"></a><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<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><br />
</div><br />
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{{:Team:UCL/Template:footerx}}</div>Ningluhttp://2014.igem.org/Team:UCL/Project/BiobricksTeam:UCL/Project/Biobricks2014-10-18T00:54:45Z<p>Ninglu: </p>
<hr />
<div>{{:Team:UCL/Template:headerx}}<br />
{{:Team:UCL/Template:BioprocessStyles}}<br />
<html><br />
<!---<br />
<a data-tip="true" class="top large" data-tip-content="TOOLTIP TEXT" href="javascript:void(0)"><b>VISIBLE TEXT</b></a><br />
---><br />
<div id="bodyContent"> <br />
<br />
<div id="TopGapO"></div><br />
<div id="BPimagewrapperHeader"><br />
<img src="https://static.igem.org/mediawiki/2014/9/98/UCLBiobricksHeaderOran.jpg" width="100%" height="auto" alt="BioBricks" /><br />
</div><br />
<br />
<div class="textArena"><br />
<!--- This is the coding for the tabs (ask sanjay before altering this) ---><br />
<ul class="tabs"><br />
<li><a href="#view1">UCL iGEM 2014</a></li><br />
<li><a href="#view2">Azoreductase</a></li><br />
<li><a href="#view3">Laccase</a></li><br />
<li><a href="#view4">Lignin Peroxidase</a></li><br />
<li><a href="#view5">Bacterial Peroxidases</a></li><br />
<li><a href="#view6">ispB asRNA</a></li><br />
<li><a href="#view7">Nuclease</a></li><br />
</ul><br />
<div class="tabcontents"><br />
<br />
<!--- This is the overview section ---><br />
<div id="view1"><div class="textTitle"><h4>Our BioBricks & how they lead to azo degradation</h4></div><br><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><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>We have strove to complete a synthetic azo dye decolourising device in E. coli which incorporates several different independent enzymes that act on azo dyes and their breakdown products to create less toxic chemicals. 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.<br />
</p><br />
<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 />
<br><br />
<p> 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.<br />
</p><b>toxicity levels</b></a>.<br />
<br><br><br />
<p> 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.<br />
To ensure that the process is entirely bio-safe we have designed a xeno-biological modules that ensures that beyond the bioreactor our organism could not survive.<br />
</p><br />
</div><br />
<br />
<!--- This is the first biobrick ---><br />
<div id="view2"><div class="textTitle"><h4>Azoreductase (BBa_K1336000)</h4></div><br><br />
<!-- This is the biobrick image --><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." 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><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>This enzyme was isolated from Bacillus subtilis, although it is also found in other bacterial species. It<br />
starts the degradation of azo dyes by reductively cleaving the azo bond.<br />
</p><br />
<p>This azo cleavage, does not only occur with azo dyes, but also with other molecules like Sulfasalazine. We isolated this enzyme from B. subtilis and converted it to BioBrick format via polymerase chain reaction (PCR). However the site directed mutagenesis did not successfully remove the illegal restriction sites and therefore we could not characterise the effects.<br />
</p><br />
<div class="textTitle"><h4>Azoreductase 1B6 (BBa_K1336001)</h4></div><br><br />
<!-- This is the biobrick image --><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." 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><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>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.<br />
</p><br />
<br />
<p>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.<br />
<p> <br />
<br />
<p>AzoR creates two or more aromatic amines (dependent on the number of azo bonds in the molecule) which are carcinogens. This is therefore only the first step of degradation. Some useful aromatic amines will be filtered off and sold as feedstock products for the fragrance industry which is further explained in the bioprocessing pages.<br />
</p> <br />
<br />
<img src="https://static.igem.org/mediawiki/2014/0/0b/SSAzoReductase.png"><br />
<br><br />
</div><br />
<br />
<!--- This is the second biobrick ---><br />
<div id="view3"><div class="textTitle"><h4>Spore Coat Protein Laccase (BBa_K1336002)</h4></div><br><br />
<!-- This is the biobrick image --><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." href="javascript:void(0)" style="width: 30%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/f/f6/UCL_BBa_K1336002.png" style="max-width: 100%;"></a><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<p>The laccase enzyme is a very non-specific oxidising enzyme. We intend to use it in concert with and as a second step to the azo reductase action to create no toxic products. Laccase will:<br />
<ul><br />
<li><br />
Break down azo bonds in specific azo dyes to non-toxic compounds <br />
</li><br />
<li>Polymerise non-specific azoreductase breakdown products to filterable, non toxic compounds<br />
</li><br />
<li>Oxidise specific azoreductase breakdown products to non-toxic compounds.<br />
</li><br />
</ul><br />
<p>The spanning range of reactions that take place in laccase are largely due to the unspecific copper mediated oxidising active site. Known reaction products are below, however these are known to break down further in ways not yet tested via NMR. <br />
</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/4e/SSlaccase1.png"><br />
<img src="https://static.igem.org/mediawiki/2014/e/eb/SSLaccase2.png"><br />
<p>Despite laccase’s unspecific active site, it cannot break down sulphonated dyes and hence oxidation of those must be left for the peroxidases (see next tab.)<br />
</p><br><br />
<br />
<br><br />
</div><br />
<br />
<!--- This is the third biobrick ---><br />
<div id="view4"><div class="textTitle"><h4>Lignin Peroxidase (BBa_K500000)</h4></div><br><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." href="javascript:void(0)" style="width: 30%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/f/f1/UCL_BBa_K500000.png" style="max-width: 100%;"></a><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<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><br />
</div><br />
<br />
<!--- This is the fourth biobrick ---><br />
<div id="view5"><div class="textTitle"><h4><em>Bacillus subtilis</em> dye-decolorizing peroxidase (BsDyP) (BBa_K1336003)</h4></div><br><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." 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><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<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 />
<div class="textTitle"><h4><em>Pseudomonas putida</em> MET94 dye-decolorizing peroxidase (PpDyP) (BBa_K1336004)</h4></div><br><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." 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><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<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><br />
</div><br />
<br />
<!--- This is the fifth biobrick ---><br />
<div id="view6"><div class="textTitle"><h4>Octaprenyl Diphosphate Synthase (ispB) (BBa_K1336005)</h4></div><br><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." 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><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<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><br />
</div><br />
<br />
<!--- This is the fifth biobrick ---><br />
<div id="view7"><div class="textTitle"><h4>Extracellular Nuclease (nucB) (BBa_K729004)</h4></div><br><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." href="javascript:void(0)" style="width: 30%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/e/ec/UCL_BBa_K729004.png" style="max-width: 100%;"></a><br />
<!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--><br />
<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><br />
</div><br />
<br />
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{{:Team:UCL/Template:footerx}}</div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-52.jpgFile:UCMCdebate (1 of 1)-52.jpg2014-09-25T18:06:18Z<p>Ninglu: </p>
<hr />
<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-51.jpgFile:UCMCdebate (1 of 1)-51.jpg2014-09-25T18:05:54Z<p>Ninglu: </p>
<hr />
<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-50.jpgFile:UCMCdebate (1 of 1)-50.jpg2014-09-25T18:05:24Z<p>Ninglu: </p>
<hr />
<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-49.jpgFile:UCMCdebate (1 of 1)-49.jpg2014-09-25T18:04:56Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-48.jpgFile:UCMCdebate (1 of 1)-48.jpg2014-09-25T18:04:50Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-46.jpgFile:UCMCdebate (1 of 1)-46.jpg2014-09-25T18:04:28Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-45.jpgFile:UCMCdebate (1 of 1)-45.jpg2014-09-25T18:04:02Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-47.jpgFile:UCMCdebate (1 of 1)-47.jpg2014-09-25T18:03:47Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-44.jpgFile:UCMCdebate (1 of 1)-44.jpg2014-09-25T18:03:20Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-43.jpgFile:UCMCdebate (1 of 1)-43.jpg2014-09-25T18:02:48Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-42.jpgFile:UCMCdebate (1 of 1)-42.jpg2014-09-25T18:02:45Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-41.jpgFile:UCMCdebate (1 of 1)-41.jpg2014-09-25T18:01:56Z<p>Ninglu: uploaded a new version of &quot;File:UCMCdebate (1 of 1)-41.jpg&quot;</p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-41.jpgFile:UCMCdebate (1 of 1)-41.jpg2014-09-25T18:01:40Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-40.jpgFile:UCMCdebate (1 of 1)-40.jpg2014-09-25T18:01:24Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-39.jpgFile:UCMCdebate (1 of 1)-39.jpg2014-09-25T18:01:11Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-37.jpgFile:UCMCdebate (1 of 1)-37.jpg2014-09-25T18:00:37Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-38.jpgFile:UCMCdebate (1 of 1)-38.jpg2014-09-25T18:00:35Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-36.jpgFile:UCMCdebate (1 of 1)-36.jpg2014-09-25T17:59:51Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-35.jpgFile:UCMCdebate (1 of 1)-35.jpg2014-09-25T17:59:36Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-34.jpgFile:UCMCdebate (1 of 1)-34.jpg2014-09-25T17:59:01Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-33.jpgFile:UCMCdebate (1 of 1)-33.jpg2014-09-25T17:58:57Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-31.jpgFile:UCMCdebate (1 of 1)-31.jpg2014-09-25T17:58:30Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-32.jpgFile:UCMCdebate (1 of 1)-32.jpg2014-09-25T17:58:12Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-30.jpgFile:UCMCdebate (1 of 1)-30.jpg2014-09-25T17:58:05Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-29.jpgFile:UCMCdebate (1 of 1)-29.jpg2014-09-25T17:57:39Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-28.jpgFile:UCMCdebate (1 of 1)-28.jpg2014-09-25T17:57:36Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-27.jpgFile:UCMCdebate (1 of 1)-27.jpg2014-09-25T17:56:47Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-25.jpgFile:UCMCdebate (1 of 1)-25.jpg2014-09-25T17:56:25Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-26.jpgFile:UCMCdebate (1 of 1)-26.jpg2014-09-25T17:56:21Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-24.jpgFile:UCMCdebate (1 of 1)-24.jpg2014-09-25T17:55:56Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-23.jpgFile:UCMCdebate (1 of 1)-23.jpg2014-09-25T17:55:39Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-22.jpgFile:UCMCdebate (1 of 1)-22.jpg2014-09-25T17:55:24Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-21.jpgFile:UCMCdebate (1 of 1)-21.jpg2014-09-25T17:55:06Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-20.jpgFile:UCMCdebate (1 of 1)-20.jpg2014-09-25T17:54:53Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-19.jpgFile:UCMCdebate (1 of 1)-19.jpg2014-09-25T17:54:44Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-18.jpgFile:UCMCdebate (1 of 1)-18.jpg2014-09-25T17:53:53Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-17.jpgFile:UCMCdebate (1 of 1)-17.jpg2014-09-25T17:53:43Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-16.jpgFile:UCMCdebate (1 of 1)-16.jpg2014-09-25T17:52:57Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-14.jpgFile:UCMCdebate (1 of 1)-14.jpg2014-09-25T17:52:34Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-15.jpgFile:UCMCdebate (1 of 1)-15.jpg2014-09-25T17:52:31Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-13.jpgFile:UCMCdebate (1 of 1)-13.jpg2014-09-25T17:51:52Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-12.jpgFile:UCMCdebate (1 of 1)-12.jpg2014-09-25T17:51:49Z<p>Ninglu: </p>
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<div></div>Ningluhttp://2014.igem.org/File:UCMCdebate_(1_of_1)-7.jpgFile:UCMCdebate (1 of 1)-7.jpg2014-09-25T17:51:00Z<p>Ninglu: </p>
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<div></div>Ninglu