Team:Bielefeld-Germany/Project/Porins
From 2013.igem.org
Porins
Overview - Porin OprF
The Microbial Fuel Cell (MFC) can be a future environmental friendly biotechnological application for the production of electrical energy. As a future alternative energy source, the bioelectricity generation must become more efficient. A major limiting factor is the low bacterial membrane permeability, hindering transport of electron shuttles through the membrane and thereby restricting the electron shuttle-mediated extracellular electron transfer (EET) from bacteria to electrodes. This results in a reduced electrical power output of the MFC. Therefore, we heterologously expressed the porin protein OprF from Pseudomonas fluorescens into Escherichia coli. This leads to a much higher current output in comparison to its parental strain (E. coli KRX). This is most likely caused by improved electron shuttle-mediated extracellular electron transfer through a dramatically increased membrane permeability. The heterologous expression of the outer membrane porin OprF from Pseudomonas fluorescens in Escherichia coli is a great genetic strategy to overcome limitations due to the membrane and to increase electricity generation by microorganisms.
Theory
The efficiency of extracellular electron transfer is a major limiting factor for electricity power output of MFCs. The electron shuttle-mediated EET is the most common EET pathway for microorganisms in a Microbial Fuel Cell such as Escherichia coli (Logan, 2009).
The cell membrane is a natural protective layer enabling proper physiology of bacteria, but it is also a barrier for substrate exchange, because an efficient electron shuttle-mediated EET requires diffusion of shuttle molecules across the cell membrane (Figure 1). However, the bacterial outer membrane is a low permeable barrier for the transport of electron shuttles across the cell membrane, which limits among other things the efficiency of electron transport and is responsible for the low power output of MFCs up to now. One strategy to improve the electron shuttle mediated EET is to enhance the permeability of the cell membrane. Evolutionary strategies, e.g. application of a continuous cell stress, are very time-consuming and unpredictable. The chemical treatment with permeabilizers can perforate the outer membrane but has negative impact on the viability and metabolism of the cells. (Liu et al., 2012)
We thus hypothesize that genetic engineering of a highly permeable cell membrane would be beneficial. Therefore, we introduce pore forming proteins in the outer membrane. These so called porins are common bacterial outer membrane proteins, which can form water-filled channels across the membrane of Gram-negative bacteria. They allow hydrophilic substances to diffuse across the outer membrane (Hancock and Brinkman, 2002).
E. coli expresses different porines, for example OmpF and OmpC. But these natural occurring porines are only permeable for molecules smaller than 600 Da, which decreases the range of usable mediators and the mediator transport kinetic (Yong et al., 2013). Thus, we thought about enhancing the amount and size of pores in the outer cell membrane with much larger porins than these of E. coli.
OprF is a major outer membrane protein in Pseudomonas species, which is a non-specific porin protein and adhesin. OprF presents one of the largest pore sizes on bacterial outer membrane that allows the diffusion of polysaccharides in a range of 2000 up to 3000 Da. In contrast to OprF the general porin channels of E. coli are only permeable to sugars with sizes smaller than 600 Da. (Yong et al., 2013)
In due consideration of all facts, heterologous expression of the porin protein OprF from Pseudomonas fluorescens in E. coli will improve the EET between bacteria and electrodes by increase of membrane permeability. A heterologous expression of large porins improves, besides mediator efficiency, the spectrum of usable mediators. Thus, alternative, environmentally friendly mediators such as NADH and riboflavin might become applicable instead of the artificial systems used up to now.
Genetic Approach
The OprF gene from Pseudomonas fluorescens was cloned and heterologously expressed in Escherichia coli KRX under the control of different promoters (Table 1).
Results
Overview
Upon the expression of the outer membrane porin protein OprF, the morphology and physicochemical characteristics of the E. coli surface were measured. SDS-PAGE combined with MALDI-TOF MS/MS, different membrane permeability assays (NPN and ONPG), a hydrophobicity assay and Atomic Force Microscopy (AFM) characterize the OprF BioBrick <bbpart>BBa_K1172501</bbpart>.
SDS-PAGE and MALDI-TOF
OprF could been detected in an SDS-PAGE with a broad overexpression band. Protein isolation of the outer membrane porin OprF by release of periplasmic protein fraction from E. coli via cold osmotic shock using Cell fractionating buffer 2.3 was successful.
The SDS-PAGE shows a significantly higher protein concentration in extracts from E.coli expressing OprF from a T7 promoter (<bbpart>BBa_K1172502</bbpart>). This is most likely caused by the higher membrane permeability (shown with NPN and ONPG uptake assay), allowing an increased release of membrane proteins by 0.2 % SDS. Nevertheless, a strong overexpression band can be observed at the expected OprF size of about 36 kDa for <bbpart>BBa_K1172502</bbpart>, which is equated with a strong expression and overproduction of OprF.
Furthermore we were able to identify the overexpressed outer membrane porin (Figure. 2) with MALDI-TOF MS/MS.
Tryptic digest of the gel band thought to represent OprF and analysis with MALDI-TOF confirmed the outer membrane porin with a Mascot Score of 222 against the bacteria database.
Hydrophobicity (Hexadecane) - Assay
The heterologous expression of the outer membrane porin OprF will enhance the hydrophobicity of cell membrane. The outward-facing side groups on each of the β-strands of the OprF monomer are hydrophobic. Therefore a positive expression should be visible by an increase in hydrophobicity. The Hexadecane-Hydrophobicity-Assay is based on different affinities to hexadecane of dissimilar cell-types according to there cell surface. Hydrophobicity can be observed by specific changes in OD600 of the aqueous phase.
An increasing hydrophobicity of cell membrane changes the physicochemical properties of the cell. This could effect for example the cell-electrode interaction. Therefore, we investigated the cellular surface characteristic by comparing Escherichia coli KRX wild type with Escherichia coli KRX with <bbpart>BBa_K1172502</bbpart>, <bbpart>BBa_K1172503</bbpart>, <bbpart>BBa_K1172504</bbpart>, <bbpart>BBa_K1172505</bbpart> and <bbpart>BBa_K1172507</bbpart> (Table 2 and Figure 3).
The OprF strain shows an increasing affinity to hexadecane with increasing promotor strength in comparison to the wild type. Cells in which OprF was expressed from the T7 promotor (<bbpart>BBa_K1172502</bbpart>) showed the maximal hydrophobicity, which was three times higher than affinity to hexadecane of the wild type.
The enhanced hydrophobicity of OprF-strains indicates a successful expression of the outer membrane porin in Escherichia coli. Such an increased hydrophobicity on the outer membrane caused by the expression of OprF will lead to an increase in the cellular adhesion to the surface of the carbon anode and an enhancement of direct electron transfer from Escherichia coli to the electrode.
Membrane permeabilty assays
NPN uptake assay
Besides testing the outer membrane hydrophobicity for physicochemical characterization of the E. coli surface, we measured the membrane permeability by NPN uptake assay for outer membrane morphology characterization (Helander and Mattila-Sandholm, 2000).
NPN is a very suitable chemical for measuring the membrane permeability of cells. An increasing NPN fluorescence intensity indicates an enhanced NPN uptaking by the outer membrane and enhanced membrane permeability (Loh et al., 1984).
Figure 8 shows a higher fluorescence emission and therefore higher membrane permeability with increasing promotor strength for OprF strains in comparison to Escherichia coli KRX wild type.
Escherichia coli with heterologous expression of the outer membrane porin OprF shows more efficient NPN uptaking than the Escherichia coli KRX Wild type, suggesting an increasing membrane permeability with increasing promotor strength. E. coli with T7 promotor (<bbpart>BBa_K1172502</bbpart>) shows the maximal membrane permeability with 100% enhanced permeability in comparison to Escherichia coli KRX Wild type. The weak Anderson promoters seem unsuitable for heterologous expression.
ONPG assay
Another way of characterizing the outer cell membrane can be achieved by ONPG assay. ONPG assay measures the membrane permeability by whole-cell lactase enzyme activity (Zhou et al., 2010).
Electron shuttle-mediated electron transfer (EET) determines the transport efficiency of electron shuttles across the cell membrane. With NPN uptake assay we were able to quantify the transport efficiency of chemical molecules across the cell membrane. ONPG assay (Whole-cell β-lactase enzyme activity assay) can show furthermore the diffusion of ONPG hydrolytic product out of the cell membrane, which quantifies not only uptaking of chemical molecules, but also the hydrolytic product secretion. With ONPG assay we are able to observe diffusion processes in and out of the cell membrane.
Due the fact that heterologous expression of OprF in Escherichia coli has no impact on the expression of β-lactase, we can assume that the β-lactase activity of Escherichia coli KRX wild type and Escherichia coli KRX with OprF expression plasmid is identical. All in all, the ONPG assay provides significantly more information than the NPN uptake assay.
The ONPG hydrolysis rate by β-lactase is much higher for Escherichia coli KRX with OprF plasmids in contrast to Escherichia coli KRX wild type. Whereas we couldn’t observe any hydrolysis activity for wild type. Escherichia coli KRX with OprF plasmids shows a much faster ONPG hydrolysis rate with increasing promotor strength and a maximal rate for Escherichia coli KRX with T7 promotor (<bbpart>BBa_K1172502</bbpart>), which is 30 times higher than wild type hydrolysis rate.
In summary we can say that the heterologous expression of OprF from Pseudomonas fluorescens in Escherichia coli significantly improves the membrane permeability. NPN and ONPG assay show correlating results. Escherichia coli outer membrane permeability is enhanced with increasing promotor strength for OprF expression. The wild type shows only a weak uptake rate of chemical molecules (NPN) but no product secretion as quantified with ONPG assay. Therefore, wild type is not suitable for a usage in the MFC. Whereas membrane optimized Escherichia coli with OprF shows great diffusion processes in and out of the cellular membrane, indicating a great optimization of electron shuttle-mediated electron transfer (EET) to the anode and increasing current production.
Atomic Force Microscopy (AFM)
In addition to morphology and physicochemical characterization of the Escherichia coli outer membrane, we wanted to visualize the surface. The technique of choice for this is Atomic Force Microscopy (AFM). After cell preparation we were able to get AFM pictures of E. coli surface with the help of the working group of [http://www.physik.uni-bielefeld.de/biophysik Prof. Dr. Dario Anselmetti], with special help from [http://www.physik.uni-bielefeld.de/biophysik/mitarbeiter/walhorn.html Dr. Volker Walhorn]. Thank you very much for your help!
Microscopy of AFM glass layer after cell preparation and before AFM measurement shows the different cell properties at the same image enlargement of the microscope and the same number of cells. Escherichia coli KRX with T7 promotor (<bbpart>BBa_K1172502</bbpart>) shows a clustering of cells, whereas wild type forms a monolayer. The increased hydrophobicity of the cell surface is already visible even with an ordinary light microscope.
AFM was carried out using the [http://www.bruker.com/de/products/surface-analysis/atomic-force-microscopy/multimode-8/overview.html MultiMode® 8 AFM from Bruker]. The measurements were performed on air with ‘Tapping Mode’ and in water with ‘Peak Force Mode’.
According to AFM images, Escherichia coli KRX with OprF and T7 promotor (<bbpart>BBa_K1172502</bbpart>) shows a slightly rougher cell surface morphology in contrast to Escherichia coli KRX wild type. Outer membrane porin OprF from Pseudomonas fluorescens usually forms trimer complex on the membrane, which leads to enhanced roughness of the cell surface. (Yong et al., 2013)
The heterologous expression of porin OprF causes a slightly rougher membrane in which the morphology and thus the viability of Escherichia coli is preserved as far as possible.
Microbial Fuel Cell Measurement
All results described by different evidence indicate an increase in energy production. All these assumptions were confirmed by cultivation of Escherichia coli KRX with OprF (<bbpart>BBa_K1172502</bbpart>) in contrast to Escherichia coli KRX wild type in the Microbial Fuel Cell.
According to our assumptions, the extracellular electron transfer mediated by electron shuttles is improved in the OprF strain resulting in an increased bioelectricity output. (Figure 10 and 11)
Escherichia coli KRX with OprF (<bbpart>BBa_K1172502</bbpart>) shows 108 % higher maximal voltage than Escherichia coli KRX wild type. Over the whole cultivation, voltage was about 100 % improved with maximum at 308 mV. Tests of M9-medium with and without mediator New Methylene Blue shows no voltage, indicating that bioelectricity generation is solely due to the bacteria.
The calculation of the electric charge confirms the described results. Electric charge is equivalent to the number of transported electrons and 111 % enhanced for Escherichia coli KRX with OprF (<bbpart>BBa_K1172502</bbpart>). The maximal electric charge of 7,2 C examines that heterologous expression of outer membrane porin OprF enhances dramatically extracellular electron transfer. High membrane permeability is crucial for efficient mediator transport across the membrane and high bioelectricity generation.
Conclusion
Cell membranes are protective layers which are crucial for physiology of the bacteria. In consideration of the Microbial Fuel Cell, membrane is also a barrier which decreases mediator and thus electron exchange between bacteria and anode. With heterologous expression of the outer membrane porin OprF from Pseudomonas fluorescens in Escherichia coli we are able to enhance membrane permeability. With this optimized cell membrane surface we generate an efficient electron shuttle-mediated EET with decreased limitation of EET by membrane barrier. In contrast to a perforation of the membrane with chemicals, cell viability is maintained with OprF expression (Liu et al., 2012). Besides improvement of EET, enhanced hydrophobicity shows optimized cell adhesion to the anode for biofilm formation and direct electron transfer.
The heterologous expression of OprF is a great genetic strategy to optimize electron shuttle-mediated electron transfer as well as electricity generation in Microbial Fuel Cells. The most suitable and efficient OprF device for Escherichia coli is a combination with Rhamnose inducible T7 promotor (<bbpart>BBa_K1172502</bbpart>).
References
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