Team:UCL/Science/Model

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    <li><a href="#view1">UCL iGEM 2014</a></li>
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    <li><a href="#view2">Azoreductase</a></li>
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    <li><a href="#view3">Laccase</a></li>
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    <li><a href="#view4">Lignin Peroxidase</a></li>
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    <li><a href="#view5">Bacterial Peroxidases</a></li>
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    <li><a href="#view6">ispB asRNA</a></li>
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    <li><a href="#view7">Nuclease</a></li>
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<div id="view1"><div class="textTitle"><h4>Our BioBricks & how they lead to azo degradation</h4></div><br>
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<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="http://2014.igem.org/wiki/images/c/c9/UCLTANELPIPETTING.JPG" style="max-width: 100%;"></a>
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<p>We plan to create a complete synthetic azo dye decolourising device in <em>E. coli</em> which incorporates several different independent enzymes that act on azo dyes and their breakdown products. After evaluating their individual breakdown characteristics, we aim to investigate the potential synergistic action of these enzymes in a single synthetic <em>E. coli</em> device and design a <a data-tip="true" class="top large" data-tip-content="We developed a novel platform for industrial scale sustainable bioremediation." href="http://2014.igem.org/Team:UCL/Science/Bioprocessing"><b>bioprocess</b></a> which could be used to upscale the method to an industrial context. </p>
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<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="http://2014.igem.org/wiki/images/c/c0/UCLTANELHOLDINGBIOBRICK.jpg" style="max-width: 100%;"></a>
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In an industrial setting, these enzymes would work sequentially in a bioreactor with preset dynamic conditions. First, azoreductase will <a data-tip="true" class="top large" data-tip-content="Via a double reduction using NADPH as a cofactor." href="javascript:void(0)"><b>cleave the azo-bond (N=N)</b></a>, producing a series of highly toxic aromatic amines. Then, these compounds will be oxidised by lignin peroxidase, laccase and bacterial peroxidases, completing decolourisation and decreasing <a data-tip="true" class="top large" data-tip-content="To the point that the final products of the process are less toxic than the intact dyes themselves." href="javascript:void(0)"><b>toxicity levels</b></a>.
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The complementary action of azoreductase, lignin peroxidase, laccase, and bacterial peroxidases will be studied in order to find out the best possible approach of sequential reaction, and this core degradation module will be extrapolated to other areas such as BioArt projects and work on <a data-tip="true" class="top large" data-tip-content="Trying to set up the foundations for a synthetic ecology." href="javascript:void(0)"><b>algal-bacterial symbiosis</b></a>.<br><br><br></p>
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Revision as of 19:33, 17 October 2014

Goodbye Azodye UCL iGEM 2014

Modelling

Our BioBricks & how they lead to azo degradation


We plan to create a complete synthetic azo dye decolourising device in E. coli which incorporates several different independent enzymes that act on azo dyes and their breakdown products. After evaluating their individual breakdown characteristics, we aim to investigate the potential synergistic action of these enzymes in a single synthetic E. coli device and design a bioprocess which could be used to upscale the method to an industrial context.


In an industrial setting, these enzymes would work sequentially in a bioreactor with preset dynamic conditions. First, azoreductase will cleave the azo-bond (N=N), producing a series of highly toxic aromatic amines. Then, these compounds will be oxidised by lignin peroxidase, laccase and bacterial peroxidases, completing decolourisation and decreasing toxicity levels.

The complementary action of azoreductase, lignin peroxidase, laccase, and bacterial peroxidases will be studied in order to find out the best possible approach of sequential reaction, and this core degradation module will be extrapolated to other areas such as BioArt projects and work on algal-bacterial symbiosis.


Overview

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):


 

 

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:


 
  • Equations for pathway model.

  •  

    Using reasonable parameter values, the simulation showed that the AzoDye is degraded within two days (48 hours). This timeframe agrees with the experimental results!


     
  • Simulated timecourse data of Acid Orange AzoDye degradation by Azoreductase, Laccase and BsDyP:
  • Parameter inference

    We wanted to see which part of the pathway is the bottleneck in degrading the 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) .


     

    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.

    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 sulfonated AzoDyes over two days.


     

    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. The parameter values that gave rise to this final population are called the 'posterior distribution'. This is shown in the figure below:


     

    The distribution of values for each parameter are shown in the diagonal. Then 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. Two parameters stand out as very constricted, k17 and k18. These are the parameters of the reactions for intake (k13) and secretion (k18) of sulfonated AzoDyes by the cell. This shows that the bottleneck happens at those two points in our pathway. So if we increase the rate of intake and secretion of AzoDyes in our synthetic pathway, we could speed up the process of AzoDye degradation!

  • Posterior distribution of model parameters
  • Flux Balance Analysis

      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 obective

      Ecoli metabolism plotted in Cytoscape (Cline, 2007):
       

      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 -1 . Without Ubiquione in the system the growth rate was found to be 0 h -1 , 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.


       
      Core metabolism map used for FBA

      References

      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.

      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.

      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.

      Orth, J.D., Thiele, I. & Palsson, B.O. (2010) What is flux balance analysis? Nature Biotechnology. [Online] 28 (3), 245–248.

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