Introduction. Global challenge of energy supply CO 2 output Radioactive waste Inefficient storage in batteries
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2 Introduction Global challenge of energy supply CO 2 output Radioactive waste Inefficient storage in batteries Limited resources Toxic waste Charging time 2
3 Fuel Cells H 2 + Anode Cathode O 2 3
4 Microbial Fuel Cells Substrate Cathode Anode O 2 4
5 Advantages Fuel Cells High efficiency Energy on demand Easy scaling Microbial Fuel Cells Same as fuel cell Variety of possible substrates Renewable resources Waste 5
6 Application Already in use with mixed cultures Escherichia coli vs. Mixed culture - electron transfer + + growth - + predictability + safety - + genetic accessibility - 6
7 Application Already in use with mixed cultures Escherichia coli vs. Mixed culture + electron transfer + + growth - + predictability + safety - + genetic accessibility - 7
8 Project Levels Hardware level Constructing a Microbial Fuel Cell Making MFC technology available to the igem community Establishing standardized measurement parameters Genome level Enabling electron transfer across membrane Enabling electron transport to anode Implementing biosafety system 8
9 Fuel Cell Evolution Requirements Electron transfer Proton transfer Anaerobic 9
10 Fuel Cell Evolution Requirements Electron transfer Proton transfer Anaerobic Nitrogen aeration Easy refill 10
11 Fuel Cell Evolution Requirements Electron transfer Proton transfer Anaerobic Nitrogen aeration Easy refill Available to community Customized for igem team York 11
12 Fuel Cell Evolution Requirements Electron transfer Proton transfer Anaerobic Nitrogen aeration Easy refill Available to community Customized for igem team York Introduced reference electrode 12
13 Fuel Cell Evolution Requirements Electron transfer Proton transfer Anaerobic Nitrogen aeration Easy refill Available to community Easy to build with 3D-printer Cheap 13
14 Genome Level Multiple approaches for each problem Crossing the cell membrane Route to the electrode Porins Cytochromes Nanowires Riboflavin GldA 14
15 Anode Mediators Electron shuttles Requirements Redox properties Soluble Non-toxic Chemical mediators Methylene blue Neutral red e - e - e - e - e - e - e - 15
16 Genetic approach Riboflavin from S. oneidensis Riboflavin Expensive exogenous mediators Heterologous expression of the riboflavin synthesis gene cluster GldA Porins Biosafety Wild type E. coli KRX Riboflavin producing strain, Anderson 0.33 (weak) Riboflavin producing strain, Anderson 0.77 (strong) 16
17 Genetic approach Riboflavin from S. oneidensis Riboflavin Expensive exogenous mediators Heterologous expression of the riboflavin synthesis gene cluster GldA Fluorescence assay Porins Enhanced extracellular riboflavin concentration Biosafety Confirmed with absorbance assay, HPLC and LC/MS measurements 17
18 Genetic approach GldA from E. coli Riboflavin Overexpression of glycerol dehydrogenase (GldA) for increasing NADH production GldA NAD + + H + + 2e - GldA NADH Porins Biosafety 18
19 Genetic approach GldA from E. coli Riboflavin Overexpression of glycerol dehydrogenase (GldA) for increasing NADH production Fluorescence-based NADH assay GldA Porins Biosafety Increased NADH production 19
20 Genetic approach GldA from E. coli Riboflavin Overexpression of glycerol dehydrogenase (GldA) for increasing NADH production Examination of increased electricity generation GldA Porins Biosafety Average electrical power increased by 40 % 20
21 Genetic approach Anode Membrane Riboflavin Membranes natural isolator GldA Optimization of mediator transport Porins Permeabilization e - e - e - e - e - Biosafety 21
22 Genetic approach OprF from P. fluorescens Riboflavin Heterologous expression of the outer membrane porin OprF One of the largest porins on bacterial outer membranes GldA Visualization via Atomic Force Microscopy Porins Biosafety Wild type E. coli KRX OprF producing strain AFM was carried out in cooperation with the Physics Department of Bielefeld University 22
23 Genetic approach OprF from P. fluorescens Riboflavin Membrane permeability assay with 1-N-phenylnaphthylamine uptake assay GldA Porins Biosafety Increased membrane permeability 23
24 Genetic approach OprF from P. fluorescens Riboflavin Electricity generation by OprF expressing strain GldA Porins Biosafety Measurements at 200 Ω resistance with methylene blue added 100 % enhanced maximal voltage 24
25 Genetic approach OprF from P. fluorescens Riboflavin Electricity generation by OprF expressing strain GldA Porins Biosafety Measurements at 200 Ω resistance with methylene blue added 400 % enhanced average electric power 25
26 Genetic approach OprF from P. fluorescens Riboflavin Electricity generation by OprF expressing strain GldA Porins Biosafety Increased membrane permeability Enabling of efficient electricity generation Further optimization possible 26
27 Genetic approach Biosafety Riboflavin GldA Porins Novel Biosafety approach inspired by igem-team Paris Bettencourt 2012 Basis: Biosafety strain D-alanine auxotrophic E. coli (Δalr ΔdadX) Complementation with safety plasmid carrying alr Additional kill switch: toxic gene product RNase Ba (Barnase) Three systems with different regulatory mechanisms AraCtive Biosafety TetOR Alive Lac of Growth 27
28 Genetic approach System AraCtive Riboflavin Rhamnose D-alanine GldA prha arac alr pbad RNase Ba Porins Biosafety 28
29 Genetic approach System AraCtive Riboflavin D-alanine GldA prha arac alr pbad RNase Ba Porins Biosafety Test with GFP instead of Barnase Functionality indirectly proven System works as expected 29
30 Achievements Design, construction and improvement of a MFC Printing a biodegradable DIY-MFC Integration of porins Riboflavin overproduction Overexpression of glycerol dehydrogenase Three advantageous biosafety systems 30
31 Achievements Human Practice Conventions SynBioDay Student Academy Media Experts Dr. Falk Harnisch Dr. Arnold Sauter Aeneas Wanner 31
32 We want to thank Prof. Dr. Jörn Kalinowski Dr. Christian Rückert Nils Lübke Timo Wolf Working groups: - Fermentation Technology - Microbial Genomics and Biotechnology 32
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