Team:Bielefeld-CeBiTec/Results/rMFC/ElectronTransfer

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        <h6>Creation of an electroautotrophic strain</h6>
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  <h6>PCR analysis</h6>
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Revision as of 15:14, 15 October 2014



rMFC

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Creation of an electroautotrophic strain

Short summary

PCR analysis

Sequencing

Phenotypic characterization with Biolog® system

Fumarate reductase

Short summary

Upon the expression of fumarate reductase in E. coli we analyzed the metabolic behavior under aerobic and anaerobic conditions. Furthermore we characterize the impact on electrochemical behavior of E. coli. We show expression of fumarate reductase frd (BBa_1465102) using SDS-PAGE in combination with MALDI-TOF/MS. Activity could be shown with HPLC analysis of fumarate consumption and succinate production. Furthermore we investigated fumarate reductase activity in different E. coli strains by phenotypic MicroArray (PM) analysis with a Biolog® system.

SDS-PAGE

The fumarate reductase could be detected in purified membrane and periplasmatic protein fraction. Proteins were fractioned by cold osmotic shock of E. coli KRX at different steps after induction of protein expression. SDS-PAGE shows the expression of fumarate reductase in E. coli KRX under control of T7 promotor (BBa_1465102). Fumarate reductase consist of four subunits, two large catalytic and two smaller membrane associated subunits (Iverson et al., 1999). The two catalytic subunits could be detected via SDS-PAGE.

Anaerobic cultivation

We cultivate E. coli KRX under anaerobic conditions to characterize activity of fumarate reductase frd (BBa_1465102 under controll of T7 promotor. Fumarate and succinate concentrations were detected with HPLC.

Phenotypic characterization with Biolog® system

The Biolog system allows Phenotype MicroArrays (PM) to analyze cellular phenotypes all at once. It is possible to characterize the influence of a lot of different energy or carbon sources, trace elements, other supplements or toxins on bacterial cells. As well as gram positive and gram negative bacteria are able to use. We tested the influence of fumarate on Escherichia coli KRX wild type and different genetically modified E. coli cells. To show activity of over expressed fumarate reductase (BBa_1465102) under controll of T7 promotor, we investigated behavior of E. coli KRX wildtype, E. coli KRX with BBa_1465102 and KRX ΔdcuB::oprF with BBa_1465102 incubated with fumarate in Biolog system.

Neutral Red

Short summary

We tested neutral red as a mediator for electron transfer into bacterial cells. The electrochemical behavior of neutral red was analyzed in a H-cell reactor.

Bromphenol Blue

Cytochroms



Reference

  • Iverson et al., 1999. Structure of the Escherichia coli Fumarate Reductase Respiratory Complex. Science, vol. 284, pp. 1961-1966

    • Retrieved from "http://2014.igem.org/Team:Bielefeld-CeBiTec/Results/rMFC/ElectronTransfer"