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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*<bbpart>BBa_K1172501</bbpart> – OprF Outer membrane porin from ''Pseudomonas fluorescens''
 
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===SDS-PAGE and MALDI-TOF===
===SDS-PAGE and MALDI-TOF===
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*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.
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[[Image:IGEM_Bielefeld_SDS_PAGE_OprF3.jpg|300px|thumb|left|<p align="justify"> '''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. '''</p>]]
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*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.
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*Furthermore we were able to identify the overexpressed outer membrane porin (Figure. 2) with MALDI-TOF MS/MS.
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**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.
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===Hydrophobicity (Hexadecane) - Assay===
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Revision as of 08:39, 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

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