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

From 2014.igem.org

(Difference between revisions)

Revision as of 09:35, 16 October 2014

Context

click to return to the Module Electron Transport

To facilitate extracellular ELECTRON TRANSPORT in E. coli we genetically introduced a heterologous electron transport pathway of the metal-reducing bacterium Shewanella oneidensis. The electron transfer pathway of S. oneidensis 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:

CymA: an inner membrane cytochrome
MtrA: a periplasmic decaheme cytochrome
MtrC: outer membrane decaheme cytochrome
MtrB: an outer membrane β-barrel protein

Figure 1. **Major components of the *S. oneidensis* electron transfer pathway**. 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 *E. coli* cytochrome with comparable functionality to CymA. Source image: adapted from Goldbeck et al. (2013).

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 Deterministic Model of EET Complex Assembly.

Jensen et al. (2010) have described a genetic strategy by which E. coli 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 S. oneidensis [3]. However, bacteria expressing the Mtr electron conduit showed impaired cell growth. To improve extracellular electron transfer in E. coli, Goldbeck et al. used an E. coli 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 mtr- and ccm 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].

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 mtrCAB under control of an weakened T7 promoter and the ccm cluster under control of the pFAB640 promoter, a combination that was found to generate the largest maximal current [2].

Improved part

E. coli strains expressing the extracellular electron transfer complex display limited control of MtrCAB expression. In addtion, these strains show impaired cell growth [2]. Use of a weakened T7 promoter upstream of the mtrCAB cluster was shown to optimize the MtrCAB expression and reduce morphological perturbations [2]. Therefore we aimed to improve the mtrCAB BioBrick of the Bielefeld 2013 team, by replacing their promoter. While characterizing their BioBrick, we could not detect the coding sequence of mtrCAB by restriction analysis or sequencing. Therefore we started from scratch to clone the mtrCAB genes under control of the weakened T7 promoter. We confirmed the sequence, and were able to show extracellular electron transport using our own mtrCAB BioBrick BBa_K1316012.

References

1.Yang et al., Bacterial extracellular electron transfer in bioelectrochemical systems. Process Biochemistry 47, 707–1714 (2012)
2. C.P. Goldbeck et al., Tuning promoter strengths for improved synthesis and function of electron conduits in E. coli ACS Synth. Biol. 2 (3), pp 150–159 (2013)
3. H.M. Jenssen et al., Engineering of a synthetic electron conduit in living cells. PNAS ∣ vol. 107 no. 45 (2010)

@@ Line 51: / Line 51: @@
 <p>
 <h3>Improved part </h3>
-<i>E. coli</i> strains expressing the extracellular electron transfer complex display limited control of MtrCAB expression. In addtion, these strains show impaired cell growth [2].  Use of a weakened T7 promoter upstream of the <i>mtrCAB</i> cluster was shown to optimize the MtrCAB expression and reduce morphological perturbations [2]. Therefore we aimed to improve the mtrCAB BioBrick of the Bielefeld 2013 team, by replacing their promoter. While characterizing their BioBrick, we could not detect the coding sequence of <i> mtrCAB </i> by restriction analysis or sequencing.  Therefore we started from scratch to clone the <i>mtrCAB</i> genes under control of the weakened T7 promoter. We confirmed the sequence, and were able to show extracellular electron transport using our own <i>mtrCAB</i> BioBrick.
+<i>E. coli</i> strains expressing the extracellular electron transfer complex display limited control of MtrCAB expression. In addtion, these strains show impaired cell growth [2].  Use of a weakened T7 promoter upstream of the <i>mtrCAB</i> cluster was shown to optimize the MtrCAB expression and reduce morphological perturbations [2]. Therefore we aimed to improve the mtrCAB BioBrick of the Bielefeld 2013 team, by replacing their promoter. While characterizing their BioBrick, we could not detect the coding sequence of <i> mtrCAB </i> by restriction analysis or sequencing.  Therefore we started from scratch to clone the <i>mtrCAB</i> genes under control of the weakened T7 promoter. We confirmed the sequence, and were able to show extracellular electron transport using our own <i>mtrCAB</i> BioBrick <a href=" http://parts.igem.org/Part:BBa_K1316012"> BBa_K1316012</a>.
 </i>