Team:Bielefeld-Germany/Data Page

From 2011.igem.org

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 sections.

How our System works

Figure 1: Production of the S-Layer fusion proteins in E. coli. To build a cellfree 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 by 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 transfered by reductase (Red), ferredoxin (Fd) and cytochrome P450. The molecular beacon (hairpin structure) binds two short DNA oligos. The NAD⁺-dependent ligase (LigA) 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-layer's ability to self-assemble on surfaces.
2. [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.
3. [http://partsregistry.org/Part:BBa_K525710 Main Page] - NAD⁺-dependent DNA ligase from E. coli : This enzyme enables determination of NAD⁺ even in very low concentrations by coupling it with a molecular beacon based assay.

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've 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-layer's 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>.