Team:Bielefeld-Germany/Project/Porins

From 2013.igem.org

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[[Image:Table1_Overview_OprF_Devices.jpg|300px|thumb|left|<p align="justify"> '''Table 1: Overview of OprF devices. Combination of OprF coding BioBrick (<bbpart>BBa_K1172502</bbpart>) with different promotors and RBS. '''</p>]]
[[Image:Table1_Overview_OprF_Devices.jpg|300px|thumb|left|<p align="justify"> '''Table 1: Overview of OprF devices. Combination of OprF coding BioBrick (<bbpart>BBa_K1172502</bbpart>) with different promotors and RBS. '''</p>]]
*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>.
*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>.
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[[Image:IGEM_Bielefeld_PSB1C3_OprF.jpg|300px|thumb|left|<p align="justify"> '''Figure 1: pSB1C3 – <bbpart>BBa_K1172501</bbpart> OprF BioBrick (1298 bp) was examined by restriction analysis and sequencing. '''</p>]]
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[[Image:IGEM_Bielefeld_PSB1C3_OprF.jpg|300px|thumb|left|<p align="justify"> '''Figure 1: pSB1C3 – <bbpart>BBa_K1172501</bbpart> OprF BioBrick (1298 bp) was examined by restriction analysis and sequencing. '''</p>]]
 
===SDS-PAGE and MALDI-TOF===
===SDS-PAGE and MALDI-TOF===
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[[Image:IGEM_Bielefeld_Figure9_Results_ONPG2.jpg|300px|thumb|left|<p align="justify"> '''Figure 5: Results of the ONPG-uptake-assay. Comparison of ONPG hydrolysis 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>. Absorbance at 405 nm wavelength with standard deviation is shown. '''</p>]]
[[Image:IGEM_Bielefeld_Figure9_Results_ONPG2.jpg|300px|thumb|left|<p align="justify"> '''Figure 5: Results of the ONPG-uptake-assay. Comparison of ONPG hydrolysis 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>. Absorbance at 405 nm wavelength with standard deviation is shown. '''</p>]]
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===Atomic Force Microscopy (AFM)===
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===Conclusion===
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Revision as of 09:00, 29 September 2013






Porins - Overview

By heterologous expression of the pore-forming transmembrane-protein (or porine) OprF from Pseudomona fluorescens a further optimization of the E. coli membrane will be achieved. E. coli possesses several own porines, for example OmpF and OmpC. But these naturally occurring porines are only permeable for molecules smaller than 600 Da, which decreases the range of usable mediators significantly. Opposed to that, porine OprF from P. fluorescence forms one of the biggest known pores in the outer bacterial membrane with a permeability of up to 3000 Da, thus improving the electron shuttle mediated extracellular electron transfer (ETT) and enabling the usage of alternative mediators such as riboflavin (vitamine B2).

Theory

Genetic Approach

Results


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.


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 wildtyp.

Figure 4: Results of the NPN-uptake-assay. Comparison of fluorescence emission 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>. Excitation at 355 nm with fluorescence emission scan from 320 up to 390 nm wavelength and with standard deviation.

  • Escherichia coli with heterologous expression of the outer membrane porin OprF shows more efficient NPN uptaking than the Escherichia coli KRX Wildtyp, 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 Wildtyp. The weak Anderson promoters seem unsuitable for heterologous expression.


ONPG assay

Figure 5: Results of the ONPG-uptake-assay. Comparison of ONPG hydrolysis 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>. Absorbance at 405 nm wavelength with standard deviation is shown.


Atomic Force Microscopy (AFM)

Conclusion

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