2

Introduction

• Global challenge of energy supply

• CO2 output

• Radioactive waste

• Inefficient storage in batteries • Limited resources

• Toxic waste

• Charging time

3

Fuel Cells

H2

+

+ + + -

+

An

od

e

Cath

od

e

+ O2

Microbial

4

Fuel Cells

+ + + -

Substrate

+

An

od

e

Cath

od

e

+ O2

Microbial

5

Advantages

Microbial Fuel Cells

• Same as fuel cell

• Variety of possible substrates

• Renewable resources

• Waste

Fuel Cells

• High efficiency

• Energy on demand

• Easy scaling

6

Application

• Already in use with mixed cultures

Escherichia coli vs. Mixed culture

electron transfer +

+ growth -

+ predictability + safety -

+ genetic accessibility -

-

7

Application

• Already in use with mixed cultures

Escherichia coli vs. Mixed culture

electron transfer +

+ growth -

+ predictability + safety -

+ genetic accessibility -

+

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

9

Fuel Cell Evolution

Requirements

Electron transfer Proton transfer □ Anaerobic

10

Fuel Cell Evolution

Requirements

Electron transfer Proton transfer Anaerobic □ Nitrogen

aeration □ Easy refill

11

Fuel Cell Evolution

Requirements

Electron transfer Proton transfer Anaerobic Nitrogen aeration Easy refill □ Available to

community

Customized for iGEM team York

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

13

Fuel Cell Evolution

Requirements

Electron transfer Proton transfer Anaerobic Nitrogen aeration Easy refill Available to

community Easy to build with

3D-printer Cheap

14

Genome Level

Multiple approaches for each problem

Riboflavin Porins Cyto-

chromes Nanowires GldA

Crossing the cell membrane

Route to the electrode

15

Mediators

• Electron shuttles

• Requirements • Redox properties

• Soluble

• Non-toxic

• Chemical mediators • Methylene blue

• Neutral red

e-

e-

e-

e-

e- e-

e-

An

od

e

16

Riboflavin from S. oneidensis

• Expensive exogenous mediators

• Heterologous expression of the riboflavin synthesis gene cluster

Wild type E. coli KRX Riboflavin producing strain, Anderson 0.33 (weak)

Riboflavin producing strain, Anderson 0.77 (strong)

Riboflavin

GldA

Porins

Biosafety

Gen

etic

ap

pro

ach

17

Riboflavin from S. oneidensis

• Expensive exogenous mediators

• Heterologous expression of the riboflavin synthesis gene cluster

• Enhanced extracellular riboflavin concentration

• Confirmed with absorbance assay, HPLC and LC/MS measurements

Fluorescence assay

Riboflavin

GldA

Porins

Biosafety

Gen

etic

ap

pro

ach

18

GldA from E. coli

• Overexpression of glycerol dehydrogenase (GldA) for increasing NADH production

NAD+ + H+ + 2e- NADH

GldA

Gen

etic

ap

pro

ach

Riboflavin

GldA

Porins

Biosafety

19

GldA from E. coli

• Overexpression of glycerol dehydrogenase (GldA) for increasing NADH production

• Fluorescence-based NADH assay

• Increased NADH production

Gen

etic

ap

pro

ach

Riboflavin

GldA

Porins

Biosafety

20

GldA from E. coli

• Overexpression of glycerol dehydrogenase (GldA) for increasing NADH production

• Average electrical power increased by 40 %

Examination of increased electricity generation

Gen

etic

ap

pro

ach

Riboflavin

GldA

Porins

Biosafety

21

Membrane

e-

e-

e-

e-

e-

An

od

e

• Membranes natural isolator

• Optimization of mediator transport

• Permeabilization

Gen

etic

ap

pro

ach

Riboflavin

GldA

Porins

Biosafety

22

OprF from P. fluorescens

• Heterologous expression of the outer membrane porin OprF

• One of the largest porins on bacterial outer membranes

Wild type E. coli KRX OprF producing strain

• Visualization via Atomic Force Microscopy

AFM was carried out in cooperation with the Physics Department of Bielefeld University

Gen

etic

ap

pro

ach

Riboflavin

GldA

Porins

Biosafety

23

OprF from P. fluorescens G

enet

ic a

pp

roac

h

• Membrane permeability assay with 1-N-phenylnaphthylamine uptake assay

• Increased membrane permeability

Riboflavin

GldA

Porins

Biosafety

24

OprF from P. fluorescens

• Electricity generation by OprF expressing strain

• 100 % enhanced maximal voltage

Measurements at 200 Ω resistance with methylene blue added

Gen

etic

ap

pro

ach

Riboflavin

GldA

Porins

Biosafety

25

OprF from P. fluorescens

• Electricity generation by OprF expressing strain

• 400 % enhanced average electric power

Measurements at 200 Ω resistance with methylene blue added

Gen

etic

ap

pro

ach

Riboflavin

GldA

Porins

Biosafety

26

OprF from P. fluorescens

• Electricity generation by OprF expressing strain

Gen

etic

ap

pro

ach

• Increased membrane permeability

• Enabling of efficient electricity generation

• Further optimization possible

Riboflavin

GldA

Porins

Biosafety

27

Biosafety

• 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

Lac of Growth

AraCtive

TetOR Alive

Gen

etic

ap

pro

ach

Riboflavin

GldA

Porins

Biosafety

28

pBAD pRha araC alr RNase Ba

D-alanine

Rhamnose

Gen

etic

ap

pro

ach

System AraCtive

Riboflavin

GldA

Porins

Biosafety

29

pBAD pRha araC alr RNase Ba

D-alanine

Gen

etic

ap

pro

ach

• Test with GFP instead of Barnase • Functionality indirectly proven • System works as expected

System AraCtive

Riboflavin

GldA

Porins

Biosafety

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

31

Achievements

Human Practice

Conventions SynBioDay Student Academy Media Experts

Dr. Falk Harnisch Dr. Arnold Sauter Aeneas Wanner

32

We want to thank…

Prof. Dr. Jörn Kalinowski Dr. Christian Rückert

Nils Lübke Timo Wolf

Working groups: - Fermentation Technology

- Microbial Genomics and Biotechnology