Team:TU Delft-Leiden/Project/Life science/EET

From 2014.igem.org

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Described are the general why and how of the practices Modeling as well as Microfluidics with respect to experimental work performed in the module Electron Transport.
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<center> <h3> Contents </h3> </center>
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Integration of Modeling, Experimental Work and Microfluidics
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<li><a href="/Team:TU_Delft-Leiden/Project/Life_science/EET"><p>Module Electron Transport</p>
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work in progress
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<li><a href="/Team:TU_Delft-Leiden/Project/Life_science/EET/theory"><p>Context</p>
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To facilitate extracellular ELECTRON TRANSPORT in <i>E. coli</i> we genetically introduced a heterologous electron transport pathway of the metal-reducing bacterium <i>Shewanella oneidensis</i>. The electron transfer pathway of <i>S. oneidensis</i> is comprised of c-type cytochromes that shuttle electrons from the inside to the outside of the cell [1]. As a result, this bacterium couples the oxidation of organic matter to the reduction of insoluble metals during anaerobic respiration. There are several proteins that define the route for the electrons and thus are the major components of the electron transfer pathway (see figure 1). Our key-player proteins are:<p>
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<li><b>CymA</b>: an inner membrane cytochrome</li>
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                    <li> <a href="/Team:TU_Delft-Leiden/Project/Life_science/EET/CM_ET"><p> Carbon Metabolism and Electron Transport </p>
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<li><b>MtrA</b>: a periplasmic decaheme cytochrome</li>
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<li><b>MtrC</b>: outer membrane decaheme cytochrome</li>
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<li><b>MtrB</b>: an outer membrane β-barrel protein</li>
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<li><a href="/Team:TU_Delft-Leiden/Project/Life_science/EET/integration"><p>Integration of Departments</p>
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<li> <a href="/Team:TU_Delft-Leiden/Project/Life_science/EET/cloning"><p> Cloning </p>
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<center><img src="https://static.igem.org/mediawiki/2014/5/5a/Delft2014_Mtr_pathway.png" width="70%"></center>
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Figure 1. <b>Major components of the <i>S. oneidensis</i> electron transfer pathway</b>. Via a series of intermolecular electron transfer events, e.g. from menaquinol (QH) to CymA, from CymA to MtrA, and from MtrA via membrane pore MtrB to MtrC, the electrons find their way to the extracellular space. The electrons are derived from L-lactate oxidation by L-lactate dehydrogenase. NapC is a native <i>E. coli</i> cytochrome with comparable functionality to CymA. Source image: adapted from Goldbeck et al. (2013).
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Now we have our so called Mtr electron conduit, but it will not function unless the multiple post-translational modifications are correctly carried out. Luckily, the cytochrome C maturation (Ccm) proteins help the conduit proteins to mature properly by providing them with heme, which is one of the requirements to carry and transfer electrons [2]. The step-by-step assembly of the Mtr protein complex is described in more detail in <a href="https://2014.igem.org/Team:TU_Delft-Leiden/Modeling/EET/Deterministic">Deterministic Model of EET Complex Assembly</a>.
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Jensen et al. (2010) have described a genetic strategy by which <i>E. coli</i> was capable to move intracellular electrons, resulting from metabolic oxidation reactions, to an inorganic extracellular acceptor by reconstituting a portion of the extracellular electron transfer chain of <i>S. oneidensis</i> [3]. However, bacteria expressing the Mtr electron conduit showed impaired cell growth. To improve extracellular electron transfer in <i>E. coli</i>, Goldbeck et al. used an <i>E. coli</i> host with a more tunable expression system by using a panel of constitutive promoters. Thereby they generated a library of strains that separately transcribe the <i>mtr</i>- and <i>ccm</i> operons. Interestingly, the strain with improved cell growth and fewer morphological changes generated the largest maximal current per cfu (colony forming unit), rather than the strain with more MtrC and MtrA present [2].
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In the ELECTRON TRANSPORT module we aimed to reproduce the results reported by Goldbeck et al. in a BioBrick compatible way. To our knowledge we are the first iGEM team that successfully BioBricked the mtr pathway. On top of that, we have succeeded to BioBrick <i>mtrCAB</i> under control of an adjusted T7lac promoter and the <i>ccm</i> cluster under control of the pFAB640 promoter, a combination that was found to generate the largest maximal current [2].
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<h2> Cloning Strategy and Characterisation of this module</h2>
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  <li> <a href="/Team:TU_Delft-Leiden/Project/Life_science/EET/cloning"><p> Cloning </p>
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                    <li> <a href="/Team:TU_Delft-Leiden/Project/Life_science/EET/cloning/GoldenGate"><p> Golden Gate Assembly</p>
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  <li> <a href="/Team:TU_Delft-Leiden/Project/Life_science/EET/characterisation"><p> Characterisation </p>
  <li> <a href="/Team:TU_Delft-Leiden/Project/Life_science/EET/characterisation"><p> Characterisation </p>
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<h3> References </h3>
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1.Yang et al., Bacterial extracellular electron transfer in bioelectrochemical systems. <i>Process Biochemistry</i>  47, 707–1714 (2012)
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2. C.P. Goldbeck et al., Tuning promoter strengths for improved synthesis and function of electron conduits in E. coli<i> ACS Synth. Biol. </i> 2 (3), pp 150–159 (2013)
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3. H.M. Jenssen et al., Engineering of a synthetic electron conduit in living cells. <i> PNAS ∣</i> vol. 107 no.  45 (2010)
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Revision as of 21:04, 13 October 2014

Module ‘Electron Transport'

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