Team:CLSB-UK/Project/Porins

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Porins

Overview

A limiting factor which we identified in BPV efficiency was the low permeability of the bacterial plasma membrane to suitable redox mediators^[1]. We aimed to overcome this issue by permeabilizing the cell surface through the integration of large membrane porins. Therefore, by heterologously expressing the porin protein BBa_K1172501 (OprF) into the synechocystis bacteria we aimed for it to lead to an improved electron shuttle-mediated extracellular electron transfer; by this occurring it would allow for a greater current output in comparison to a microbial fuel cell which had the parental strain of the bacteria.^[6]

Theory

Porins are beta barrel proteins that cross a cellular membrane and act as a pore through which molecules can passively diffuse; porins allow passive diffusion to occur through the outer cell membrane acting specifically (chemically selective) for different types of molecules. They are composed of β strands, which are linked together by beta turns on the cytoplasmic side and long loops of amino acids on the other^[1]. The β strands lie in an antiparallel fashion and form a cylindrical tube, called a β barrel. The amino acid composition of the porin β strands is unique in that polar and non-polar residues alternate along them. This means that the non-polar residues face outward so as to interact with the non-polar lipid membrane, whereas the polar residues face inwards into the centre of the beta barrel to interact with the aqueous channel.^[1]

Naturally the plasma membrane acts in bacteria as a layer enabling suitable physiology to occur within the cell as well as acting as a barrier for substrate exchange but this leads to the limitation of low membrane permeability. Thus, to tackle this we will have to enhance the permeability of the cell membrane using the porin OprF.^[2]

The outer membrane porin protein OprF can be heterologously expressed from another gram negative bacteria (Porin OprF comprises of only the N-terminal, and has a 162-amino-acid domain).^[4] These porins are quite common in the outer membranes of many different gram-negative bacteria and they are able to form water filled channels across the span of the membrane. This allows for hydrophilic substances to be diffused through the outer membrane.^[3]

Synechocystis expresses different porins in its outer membrane however the natural porins tend to be permeable for molecules of size <700 Da and this severely reduces the range of usable mediators and the mediator transport kinetics.^[2] Hence, we decided to use the porin OprF to enhance the size of the pores that are present in the bacterial outer membrane. OprF is a major outer membrane protein in Pseudomonas fluorescens, acting as a non-specific porin protein and adhesin. OprF represents one of the largest pore sizes on bacterial outer membranes allowing diffusion of polysaccharides in a range of 2000 to 3000 Da. This significantly increases the spectrum of usable mediators.^[2] The outward-facing side groups on each of the β-strands of the OprF monomer are hydrophobic. Therefore, heterologous expression should be detectable by an increase in hydrophobicity.

From research we have been able to identify that by the use of the protein OprF will increase the range of mediators which we will be able to use for the MFC. By having the increased range of mediators at our disposal, we can attempt to use alternative mediators which are more eco-friendly and safe for use.^[5]

Genetic Approach

As the genetic approach for increasing the membrane permeability of our microbial fuel cell, we planned to express the gene into our bacteria; we first cloned the BBa_K1172501 part and we then heterologously expressed this foreign gene into synechocystis under the control of several different promoters. At the end of this, we had successfully been able to attach the oprF gene to our promoter and thus we aimed to use this to obtain measurements from an experimental setup.

Results

What we had aimed for from expressing this porin protein OprF was an enhancement of the hydrophobicity of the cell membrane as well as an increase in the membrane permeability thus increasing the extracellular electron transfer. Unfortunately, we were unable to find a ribosome binding site suitable for the part and for use in cyanobacteria. This meant that although we completed the assembly of the promoter and OprF, it has no functional purpose.

References

• [1] Porins at the US National Library of Medicine Medical Subject Headings (MeSH)
• [2] Niederweis, Michael (2003-09-01). "Mycobacterial porins--new channel proteins in unique outer membranes". Molecular Microbiology: 1167–1177.
• [3] Department of Molecular and Cell Biology, University of California-Berkeley, Berkeley, CA 94720-3202, USA.
• [4] Branden and Tooze, Introduction to Protein Structure, second edition.
• [5] Logan BE (2009) Exoelectrogenic bacteria that power microbial fuel cells. Nat Rev Microbiol (7): 360–387.
• [6] Yong YC, Yu YY, Yang Y, Liu J, Wang JY, Song H (2013) Enhancement of Extracellular Electron Transfer and Bioelectricity Output by Synthetic Porin. Biotechnology and Bioengineering 110 (2): 408-416.