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

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<h2> Extracellular Electron Transport Module</h2>
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    <h2>Module Electron Transport</h2>
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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 (Yang et al., 2012). 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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        Information with respect to literature consulted regarding the Modules is referred to under Context. Also, each of the three complementary Modules is equipped with an Integration of Departments, in which it is described how the Departments Modeling, Experimental Work and Microfluidics interact. Furthermore, each Module contains information on Cloning and results are presented under Characterization.
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      <ul> Module Electron Transport
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            <li><a href="/Team:TU_Delft-Leiden/Project/Life_science/EET/theory">Context</a></li>
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            <li><a href="/Team:TU_Delft-Leiden/Project/Life_science/EET/integration">Integration of Departments</a></li>
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            <li><a href="/Team:TU_Delft-Leiden/Project/Life_science/EET/cloning">Cloning</a></li>
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            <li><a href="/Team:TU_Delft-Leiden/Project/Life_science/EET/characterisation">Characterization</a></li>
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Keen to see our <b>conclusions</b> for this module?  See the list below!
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<li><b>CymA</b>: an inner membrane cytochrome</li>
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<li>The <i> mtrCAB</i> genes under control of the weakened T7lacO promoter were successfully BioBricked.</li>
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<li><b>MtrA</b>: a periplasmic decaheme cytochrome</li>
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<li>The <i> ccm </i> genes under control of the pFAB640 promoter were successfully BioBricked. </li>
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<li><b>MtrC</b>: outer membrane decaheme cytochrome</li>
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<li>Expression of both our <i>mtrCAB</i> BioBrick and <i>ccm</i> BioBrick results in Extracellular Electron Transport. </li>
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<li><b>MtrB</b>: an outer membrane β-barrel protein</li>
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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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Want to know how we came to these conclusions? Go to our <a href=https://2014.igem.org/Team:TU_Delft-Leiden/Project/Life_science/EET/characterisation><b>Characterization</b></a> page!</p>
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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>. 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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        Interested in one of our other Modules? Navigate to the&nbsp; <a href="https://2014.igem.org/Team:TU_Delft-Leiden/WetLab/landmine"> <b> Module Landmine Detection </b> </a> page and discover how we tweaked <i> E. coli</i> to let it respond on landmines. Interested in living materials? Go to our  <a href="https://2014.igem.org/Team:TU_Delft-Leiden/Project/Life_science/curli"> <b> Module Conductive Curli </b> </a> and find out how you can combine the benefits of both living- and non-living materials.
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<h2> Cloning Strategy and Characterisation of this module</h2>
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"https://static.igem.org/mediawiki/2014/8/86/Module_electron_transport_transfer_pathway_with_Mtr_protein_complex.jpg" width="100%" height="100%">
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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/characterisation"><p> Characterisation </p>
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<h3> References </h3>
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<p>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)</p>
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Figure 1: General scheme of biosensory plug-and-play E.coli cell responsive to, in this particular case, TNT. Generated is a measureable electrical current via the implemented extracellular electron transport pathway.
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Latest revision as of 20:49, 17 October 2014

Module Electron Transport

Information with respect to literature consulted regarding the Modules is referred to under Context. Also, each of the three complementary Modules is equipped with an Integration of Departments, in which it is described how the Departments Modeling, Experimental Work and Microfluidics interact. Furthermore, each Module contains information on Cloning and results are presented under Characterization.

Keen to see our conclusions for this module? See the list below!

  • The mtrCAB genes under control of the weakened T7lacO promoter were successfully BioBricked.
  • The ccm genes under control of the pFAB640 promoter were successfully BioBricked.
  • Expression of both our mtrCAB BioBrick and ccm BioBrick results in Extracellular Electron Transport.

Want to know how we came to these conclusions? Go to our Characterization page!


Interested in one of our other Modules? Navigate to the  Module Landmine Detection page and discover how we tweaked E. coli to let it respond on landmines. Interested in living materials? Go to our Module Conductive Curli and find out how you can combine the benefits of both living- and non-living materials.



Figure 1: General scheme of biosensory plug-and-play E.coli cell responsive to, in this particular case, TNT. Generated is a measureable electrical current via the implemented extracellular electron transport pathway.
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