Energy production in Escherichia coli using exoelectrogenic genes (Mtr-pathway) found in Shewanella oneidensis MR-1

Dr Mark Clements, Fapetu Segun, Thomas Bridge, Zeljka Kalinic, Amy MacLatchy, Ishwak Ahmed, Paulina Brajer, Poppy Brown, Alina Chrzastek, Nedaa Jeway, Hira Khan, Hibaq Obsiye, Nura Sharif, Shuhanaz Shuma and Amritpal Singh.

Faculty of Science and Technology, University of Westminster, London, W1W 6UW

Figure 6. The voltage-time profile showed that MFCs utilising the 408-positive control generated the highest voltage at the day 3 under 1000Ω resistor. Overtime MtrCAB demonstrated that it was almost as effective at electron transfer as the positive control 408 . MtrCAB demonstrated that it was sustainable over time as would be expected for microorganisms in a MFC.

Figure 5. An image of the Western blot demonstrating that the protein CymA has been expressed as seen by the band at the expected size of 40.5 kDa, extracted from colony number 1. Two colonies were selected (MtrC-His , MtrA-His and CymA-His). A His tag was added to the proteins MtrC, MtrA and CymA in order to determine whether the correct proteins were being produced. CymA was the only protein that is visible on the Western blot.

Results

Figure 4. Polarisation curves in order to determine the internal resistance of MFCs for MtrA, MtrC and MtrCAB cytochromes. The positive control-408 had the highest current production, followed by MtrCAB, then positive control-444. MtrA was the least productive of the MFCs measured.

Figure 3. The comparison of MFC performance during the lag phase of E.coli growth. As shown 408-positive control and 444-Positive Control gave maximum power density of 98±4mWm-2 and 67±3mWm-2 respectively. MtrCAB modified Top10 E. coli gave 89±2mWm-2; MtrC gave 70±3mWm-2 while MtrA gave 42±3mWm-2. The result of the control produced insignificantly maximum power generation of 0.98mWm-2

Anti-His tag Antibody Western blot of MtrC, MtrA and CymA proteins

‘Energy is the single most important problem facing humanity today’ as quoted by Richard Smalley, late Nobel laureate, in 2002. Hence, the reason for exploring the use of synthetic biology for modifying Escherichia coli for microbial fuel cells (MFCs). Shewanella oneidensis MR-1 is a dissimilatory metal reducing bacterium. One of the several electron transport chains found in S.oneidensis is the Mtr pathway. This specific pathway is involved in the accepting of electrons which then carries a potential electrical charge. By cloning this pathway into E. coli, the aim is to produce an efficient electric producing microbial fuel cell (MFC). The efficiency of the MFC is down to the biofilm which is formed when the cells adhere to a surface and to each other. S.oneidensis is capable of transferring electrons through extensions known as nanowire. We have explored electron transfer through the use of flagella found in E.coli K-12 derivative DH5-α.

-The Mtr pathway works as a chain of five proteins (CymA-MtrC,A,B-OmcA- Fig.1) that transfer electrons through the inner and outer membrane of the bacteria to an electron acceptor (1).

-We hope to Improve the electrical potential of a microbial fuel cell (MFC) by cloning this pathway into E.coli. Figure 1. Image of the Mtr pathway (2)

Figure 2. Microbial fuel cell

-The E.coli are grown in an anaerobic chamber of the MFC (Fig. 2) forming a biofilm on the anode.

-Electrons are striped from the carbon source, in our case wastewater, by an oxidation reaction, and are transferred to the anode.

-Electrons move towards the aerobic cathodic chamber with a higher redox potential.

-At the cathode the electrons reduce atmospheric oxygen and combine with protons that have entered the chamber via the semipermeable membrane.

Introduction

Methodology

PARTS

Applications -Potential applications for our project could be used in both developed and developing countries for industries such as textiles, paper and pulp mills, cotton mills. brewery wastewater, etc.

-Waste water could be used as the main carbon source for our synthetic E. coli, in turn reducing the level of contaminants and producing a green energy source, which will offset the heavy reliance of fossil fuels.

Conclusions and future considerations-12 BioBricks were produced and electricity was generated from our recombinant E.coli. -MtrCAB construct produced the most energy. -The Western blot showed a band that was visible at the expected size, 40.5 kDa. -It also demonstrated that the protein CymA was successfully produced in TOP 10 E.coli cells and appeared to be non toxic. -Need to sequence the results of the Western blot in order to further investigate the findings. -Several biocontainment methods were analysed , such as the use of synthetic amino acids(3) and redundancy of codon usage(4).-Biocontainment techniques were not implemented due to financial and time constraints.

References1. TerAvest, M, A., Zajdel, T, J., and Ajo-Franklin, C, M. (2014). The Mtr Pathway of Shewanella Oneidensis MR-1 Couples Substrate Utilisation to Current Production in Escherichia coli. ChemElectroChem 1: 1874-1879

2. Fredrickson,J, K., Romine,M, F., Beliaev, A, S., Auchtung,M, J., Driscoll, M, E., Gardner, T, S., Nealson, K, H., Osterman, A, L., Pinchuk, G., Reed, J, L., Rodionov, D, A., Rodrigues, J, L. M., Saffarini, D, A., Serres, M,H., Spormann, A, M., Zhulin, I, B., and Tiedje, J, M. (2008). Towards environmental systems biology of Shewanella. Nature Reviews Microbiology 6, 592-603

3. Rovner, A, J., Haimovich, A, D., Katz, S, R., Li, Z., Grome, M, W., Gassaway, B, M., Amiram, M., Patel, J, R., Gallagher, R, R., Rinehart, J., and Isaacs, F, J. (2015). Recorded Organisms engineered to depend onsynthetic amino acids. Nature 518: 89-105

4. Mandell, D, J., Lajoie, M, J., Mee, M, T., Takeuchi, R., Kuznetsov, G., Norville J, E., Gregg, C, J., Stoddard, B, L., and Church, G, M. (2015). Biocontainment of genetically modified organisms by synthetic protein design.Nature 518: 55-74

Abstract

ApplicationsPotential applications for our project could be used in both developed and developing countries for industries such as:

- Textiles - Paper and pulp mills - Cotton mills - Brewery wastewater

The waste water from such factories could be used as the main carbon source for our synthetic E. coli. In doing so will reduce the level of contaminants in the waste water and produce a green energy source, which will offset the heavy reliance of fossil fuels.

Our MFC may have the potential to be used simultaneously as a bioenergy producer and for wastewater treatment. Thus, reducing the cost of many industrial processes through the provision of bioenergy whilst reducing the level of contamination of the wastewater.

Other examples include:

- investigating different materials for the anode and cathode and the proton membrane - exploring methods and materials in order to increase the surface area of the anode - explore issues surrounding biocontainment, consider techniques other than auxotrophic methods as mentioned in Gallagher et al, 2015, Rovner et al, 2015 and Mandell et al, 2015.

ABSTRACT ‘Energy is the single most important problem facing humanity today’ as quoted by Richard Smalley, late Nobel laureate, in 2002. Hence, the reason for exploring the use of synthetic biology for modifying

Escherichia coli for microbial fuel cells (MFCs). Shewanella oneidensis MR-1 is a dissimilatory metal reducing bacterium. One of the several electron transport chains found in S.oneidensis is the Mtr pathway. This specific pathway is involved in the accepting of electrons which then carries a potential electrical charge. By cloning this pathway into E. coli, the aim is to produce an efficient electric

producing microbial fuel cell (MFC). The efficiency of the MFC is down to the biofilm which is formed when the cells adhere to a surface and to each other. S.oneidensis is capable of transferring electrons through extensions known as nanowire. We have explored electron transfer through the use of flagella found in E.coli K-12 derivative, DH5-α.