Project

Project Overview

The goal of our project is to generate electric 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 Ecolectricity, the use of E. coli for direct energy production.

Overview

Over 100 years ago, the British botanist M. C. Potter discovered electro-chemical reactions linked to anaerobic microbial degradation processes. Based on 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 electric 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 investigated different genetic approaches. Using synthetic biology, we designed 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 was to provide for an efficient electron transfer from the bacteria to the electrode. Therefore we facilitated and improved 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 increased 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.

Microbial Fuel Cell

A Microbial Fuel Cell (MFC) can be utilized for power generation through the conversion of organic and inorganic substrates by 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, thereby creating an electric current. Because the MFC-system seems to have great potential as a means to generate renewable energy, Bielefelds 2013 iGEM Team decided to develop a system which relies on genetically modified E. coli for power generation. Compared to conventional approaches using mixed cultures from the environment, the main benefits of these bacteria are their well described characteristics as well as their accessibility to directed genetic engineering. They also grow fast and are robust towards different cultivation conditions.

Glycerol dehydrogenase

Mediators are essential for the use of Escherichia coli in Microbial Fuel Cells. The main challenge 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 to overexpress the 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.

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 MFC, which made riboflavin overproduction a suitable target for optimization of our MFC.
We have shown that cloning of the riboflavin cluster from the 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

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 (PCA) against fungal root decease. 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

A major limiting factor of electron transfer to the electrode and efficient bioelectricity generation is the low bacterial membrane permeability, limiting transport of mediators 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 in Escherichia coli with great success.

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 protein MtrA, the outer membrane β-barrel protein MtrB and the outer membrane cytochrome MtrC was isolated from Shewanella oneidensis MR-1 and heterologously expressed in E. coli.

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.

Biosafety

Biosafety is an essential aspect of synthetic biology, because the living organisms could possibly get out of your containment system due to damage or incorrect handling and interact with the environment. There exist a plethora of useful systems to prevent the bacteria from escaping or killing the bacteria when they have escaped physical containment. To complement this collection we constructed not only one system but 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. Therefore, the constructed Biosafety system takes the best of this two approaches and is characterized as a double kill-switch system. Additionally, this double kill-switch mechanism provides a higher plasmid stability and a higher resistance towards undesirable mutations. In short: Our Biosafety-System is safe!