Team:Bielefeld-Germany/Project/Porins

Porins - Overview

Figure 1: Schematic of the enhancement mechanism of electron shuttle-mediated electron transfer between bacteria and the anode of MFCs by the synthetic porin OprF.

Results

Overview

• The OprF gene from Pseudomonas fluorescens was cloned and heterologously expressed in Escherichia coli KRX under the control of different promoters (Table 1).

Table 1: Overview of OprF devices. Combination of OprF coding BioBrick (<bbpart>BBa_K1172502</bbpart>) with different promotors and RBS.

• 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 OprF BioBrick <bbpart>BBa_K1172501</bbpart>.

Figure 1: pSB1C3 – <bbpart>BBa_K1172501</bbpart> OprF BioBrick (1298 bp) was examined by restriction analysis and sequencing.

SDS-PAGE and MALDI-TOF

• OprF could been detected with SDS-PAGE. Protein isolation of the outer membrane porin OprF by Release of periplasmic protein fraction from E. coli by cold osmotic shock using Cell fractionating buffer 2.3 was successful.

Figure 2: SDS-PAGE with Prestained Protein Ladder from Thermo Scientific as marker. Comparison of protein expression between Escherichia coli KRX wild type and Escherichia coli KRX with <bbpart>BBa_K1172502</bbpart>, <bbpart>BBa_K1172503</bbpart> and <bbpart>BBa_K1172507</bbpart> after periplasmic protein fractioning with Cell fractioning buffer 2.3.

• The SDS-PAGE shows a significantly higher protein concentration for E.coli with OprF and T7 promoter (<bbpart>BBa_K1172502</bbpart>). It seems to be that the higher membrane permeability (shown with NPN and ONPG uptake assay) allows a better release of membrane proteins by 0.2 % SDS. Nevertheless, we can see a strong overexpression band 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 lane for analysis with MALDI-TOF could examine the outer membrane porin with a Mascot Score of 222 against 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.
• 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 wildtyp 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 Wildtyp. OprF with T7 promotor (<bbpart>BBa_K1172502</bbpart>) shows the maximal hydrophobicity which was three times higher than affinity to hexadecane of the Wildtyp.

Table 2: Results of the Hexadecane-Hydrophobicity-Assay. Comparison of protein Hydrophobicity between Escherichia coli KRX wild type and 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>. Affinity to hexadecane (Hydrophobicity) with standard deviation and enhancement in comparison to the wild-type is shown.

Figure 3: Results of the Hexadecane-Hydrophobicity-Assay. Comparison of protein Hydrophobicity between Escherichia coli KRX wild type and 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>. Affinity to hexadecane (Hydrophobicity) with standard deviation and enhancement in comparison to the wild-type is shown.

• 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.