Team:Bielefeld-Germany/Data Page

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Revision as of 11:39, 21 September 2011

This page gives a basic overview about our cell-free bisphenol A biosensor system and the BioBricks we have used. A more detailed description of the biosensor system can be found in our project description and in the bisphenol A, S-layer and NAD⁺ detection background subsections.

How our system works

Figure 1: Production of the S-layer fusion proteins in E. coli. To build a cell-free bisphenol A biosensor the S-layer fusion proteins have to be extracted from the cells and purified (simplified schema).

Figure 2: Coating of the silica beads with the S-layer fusion proteins. Every silica bead gets covered with a geometric S-layer film consisting of an alternating structure of the two essential S-layer fusion proteins.

Figure 3: Visualization of our cell-free bisphenol A biosensor system with all essential components. Bisphenol A (BPA) is reduced by the electrons from NADH transferred by the ferredoxin-NADP⁺ oxidoreductase (FNR, <partinfo>BBa_K525499</partinfo>), ferredoxin (Fd, <partinfo>BBa_K123000</partinfo>) and cytochrome P450 (CYP, <partinfo>BBa_K123001</partinfo>) which are fused to a S-layer protein. The molecular beacon (hairpin structure) binds two short DNA oligos. The NAD⁺-dependent ligase (LigA), which is also fused to S-layer proteins, ligates the two oligos so that the hairpin structure opens up and the fluorophore is able to emit light after extinction.

Data for our favorite new parts

1. [http://partsregistry.org/Part:BBa_K525305 Main Page] - Fusion protein of S-layer SgsE and mCitrine: This fluorescent S-layer fusion protein is used to characterize purification methods and to demonstrate the S-layers ability to self-assemble on surfaces.
2. [http://partsregistry.org/Part:BBa_K525515 Main Page] - Fusion protein of BisdA and BisdB: This fusion protein improves the bisphenol A degradation in E. coli compared to the so far in the partsregistry existing BPA degrading BioBricks.
3. [http://partsregistry.org/Part:BBa_K525710 Main Page] - NAD⁺-dependent DNA ligase from E. coli (LigA) : This enzyme enables determination of NAD⁺ even in very low concentrations by introducing it to a molecular beacon based bioassay.

Data for pre-existing parts

1. [http://partsregistry.org/Part:BBa_K123000:Experience Experience] - BisdA degrades Bisphenol A when used with BisdB, BBa_K123000 (University of Alberta, iGEM 2008): Complete degradation of 120 mg L^-1 Bisphenol A with polycistronic bisdAB gene in 30-33 h. Even faster (21-24 h) when using a fusion protein of BisdA and BisdB.
2. [http://partsregistry.org/Part:BBa_K123001:Experience Experience] - BisdB degrades Bisphenol A when used with BisdA, BBa_K123001 (University of Alberta, iGEM 2008): Complete degradation of 120 mg L^-1 Bisphenol A with polycistronic bisdAB gene in 30-33 h. Even faster (21-24 h) when using a fusion protein of BisdA and BisdB.

We have also characterized the following parts

1. [http://partsregistry.org/Part:BBa_K525405 Main Page] - Fusion protein of S-layer SbpA and mCitrine: This fluorescent S-layer fusion protein is used to characterize purification methods and to demonstrate the S-layers ability to self-assemble on surfaces.
2. [http://partsregistry.org/Part:BBa_K525512 Main Page] - Polycistronic expression of BisdA and BisdB: This is the version of BPA degrading BioBricks found in the partsregistry - comparison to our fusion protein <partinfo>K525515</partinfo>.
3. [http://partsregistry.org/Part:BBa_K525517 Main Page] - Fusion Protein of BisdA and BisdB (expressed): This fusion protein improves the bisphenol A degradation in E. coli compared to the so far in the partsregistry existing BPA degrading BioBricks.
4. [http://partsregistry.org/Part:BBa_K525234 Main Page] - Fusion protein of S-layer CspB and mRFP: This fluorescent S-layer fusion protein is used to characterize the intracellular localisation as well as purification methods for CspB S-layers.