Project

Project Overview

The goal of our project is to generate electrical energy with a genetically modified ''Escherichia coli'' in a self-constructed fuel cell. Besides the design, construction and technical optimization of the fuel cell, we investigate different genetic approaches. Using synthetic biology, we are designing different BioBricks for bioelectricity generation. IGEM-Team Bielefeld 2013 enables ''E''co''lectricity'', the use of ''E. coli'' for direct energy production.

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Over 100 years ago the British botanist M. C. Potter discovered electro-chemical reactions linked to anaerobic microbial degradation processes. From this milestone sprung the first ideas for developing biological degradation systems for electricity generation. Today, there is a growing interest in the use of environmentally friendly alternative energy sources to combat the depletion of fossil fuels and an increasing pollution of the environment. Therefore, iGEM-Team Bielefeld is developing an Escherichia coli based Microbial Fuel Cell (MFC).

The goal of our project is to generate electrical energy with a genetically modified Escherichia coli in a self-constructed fuel cell. Besides the design, construction and technical optimization of the fuel cell, we investigate different genetic approaches. Using synthetic biology, we are designing different BioBricks for bioelectricity generation. Specific electron transfer proteins have been compiled from a variety of organisms, in order to gain an Escherichia coli Fuel Cell platform, which turns E. coli to an electro active organism. The main challenge is to provide for an efficient electron transfer from the bacteria to the electrode. Therefore we facilitate and improve electron donation by producing electron-shuttles, so called endogenous mediators, as well as permeabilizing the cell surface by integrating large membrane porins and providing a direct electron pathway by conductive transmembrane protein structures. All these electron transport elements increase electron transfer and bioelectricity generation.

With different aspects for technical and genetic optimization we enable Ecolectricity, the use of E. coli for direct energy production. Furthermore we formed a biosafety concept to use our Microbial Fuel Cell not only in the laboratory, but also in the future as a safe alternative energy source for numerous and manifold of electronic applications.

MFC

• A microbial fuel cell (MFC) can be utilized for power generation through the conversion of organic and inorganic substrates by the use of microorganisms. The fuel cell generally consists of two separate units, the anode and cathode compartment which are separated by a proton exchange membrane (PEM). Microorganisms, acting as biocatalysts, release electrons during metabolic reactions and transfer them to the anode of the fuel cell. The electrons pass through an external load circuit to reduce an electron acceptor located in the cathode compartment and by doing so, create an electric current. Because the MFC-system has sufficient potentials and offers an interesting approach for renewable energies production Bielefelds 2013 iGEM Team decided to develop a system which relies on genetically modified E. coli for power generation. The main benefit being that these bacteria grow fast and are quite robust regarding cultivation conditions.

Glycerol dehydrogenase

• Mediators are essential for the use of Escherichia coli in Microbial Fuel Cells. The main advantage of improving MFCs is to enhance kinetics of the electron transfer between the bacterial cells and the fuel cell anode. Enhancing the mediator concentration in the MFC is an efficient way for higher electron transfer. In order to decrease the usage of expensive and toxic synthetic mediators, we engineered an E. coli KRX strain with overexpression of glycerol dehydrogenase (GldA). GldA produces the endogenous mediator NADH from NAD⁺ and glycerol, which is the main carbon source of our medium. Optimized E. coli produces efficient mediators. Read more about our subproject GldA, of the theory behind, the genetic approach, to the results.

Riboflavin

• Riboflavin, or Vitamin B2 is a redox-active substance that plays an essential role in living cells. Secreted into the medium, it can be effectively used by some bacteria for electron transfer. Presence of riboflavin in anaerobic cultures leads to higher current flow in a microbial fuel cell, which made riboflavin overproduction a suitable target for optimisation of our MFC.
  We have shown that cloning of the riboflavin cluster from a metal-reducing bacterium Shewanella oneidensis MR-1 in E. coli is sufficient to achieve significant riboflavin overproduction detectable both in supernatant and in cells.

Phenazine

• <p align="justify">Phenazines are water-soluble secondary metabolites secreted by some bacterial species. Almost all their properties can be attributed to their ability to undergo redox transformations. Some soil-born bacteria commonly produce phenazine-1-carboxylic acid ([http://www.ebi.ac.uk/chebi/chebiOntology.do?chebiId=CHEBI:62412 PCA])) against fungal root decease. (Niknejad Kazempour, [http://www.agriculturaits.czu.cz/pdf_files/vol_42_4_pdf/kazempour.pdf 2009]) Bacteria are also able to use this substance as a mediator for electron shuttling, which leads to an efficiency gain if a phenazine-producing strain is present in a MFC.

Porins

• <p align="justify">A major limiting factor of electron transfer to the electrode and efficient bioelectricity generation is the low bacterial membrane permeability, limiting transport of electron shuttles through the membrane that restricts the electron shuttle-mediated extracellular electron transfer (EET). This results in a reduced electrical power output of the MFC. Therefore, we heterologously expressed the porin protein OprF from Pseudomonas fluorescens into Escherichia coli with great success. Read more about our subproject porins, of the theory behind, the genetic approach, to the results.

Cytochromes

• Cell membranes work as a natural insulator and prevent the flow from electrons out of the cell. To enable transfer of electrons from the general metabolism to the outside of the cell a minimal set of genes, coding for the periplasmatic decaheme MtrA, the outer membrane β-barrel protein MtrB and the outer membrane cytochromes MtrC was isolated from Shewanella oneidensis MR-1 and heterologously expressed in E. coli. Read more about our subproject cytochromes, the theory behind it, the genetic approach and the results.

Nanowires

• Multiple bacteria form special, electrically highly conductive pili, which are required for survival in anaerobic environments. Electrons, generated through the oxidation of different substrates can be transported by these pili and transferred to alternative solid electron-acceptors. These properties characterize them as an interesting option for the optimization of E. coli for the use in MFCs. Unfortunately multiple genes, arranged in large gene clusters are required to form nanowires in organisms such as Geobacter sulfurreducens, so that the planned cloning of a functional expression system seems to be a big challenge.Read more about our subproject cytochromes, the theory behind it, the genetic approach and the results.

Biosafety

• Biosafety is an essential aspect when taking part in iGEM especially when you work with living organisms which could possibly get out of your application by damage or incorrect handling. It is important to protect the population and the environment by developing and applying systems which comply these aspects. In order to counter this problem there exist useful systems to prevent the bacteria from escaping or killing the bacteria when they are outside of the application. To complement this archive we constructed not only one system but also three systems which differ in leakiness and strength. For this approach we combined two common Biosafety-ideas, an auxotrophic and a toxic gene product, in one device. So the constructed Biosafety-System takes the best of this two approaches and is characterized by a double kill-swtich system. This double kill-switch mechanism provides additional a higher plasmid stability and a higher resistance towards undesirable mutations. In one sentence: Our Biosafety-System is safe! Read more about our subproject biosafety, of the theory behind, the genetic approach, to the results.