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 as well as 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, <partinfo>BBa_K525710</partinfo>), 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. <partinfo>K525305</partinfo> - 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. <partinfo>K525515</partinfo> - 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. <partinfo>K525710</partinfo> - 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. 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. 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. <partinfo>K525405</partinfo> - 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. <partinfo>K525512</partinfo> - 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. <partinfo>K525517</partinfo> - 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. <partinfo>K525234</partinfo> - 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 from Corynebacterium halotolerans.
5. <partinfo>K525121</partinfo> - S-layer protein CspB with TAT-sequence and lipid anchor: This fluorescent S-layer fusion protein is used to characterize the intracellular localisation and purification methods for CspB S-layers from Corynebacterium glutamicum.
6. <partinfo>K525123</partinfo> - S-layer protein CspB with lipid anchor: This fluorescent S-layer fusion protein is used to characterize the intracellular localisation and purification methods for CspB S-layers from Corynebacterium glutamicum.
7. <partinfo>K525222</partinfo> - S-layer protein CspB: This fluorescent S-layer fusion protein is used to characterize the intracellular localisation and purification methods for CspB S-layers from Corynebacterium halotolerans.
8. <partinfo>BBa_K525223</partinfo> - S-layer protein CspB with lipid anchor: This fluorescent S-layer fusion protein is used to characterize the intracellular localisation and purification methods for CspB S-layers from Corynebacterium halotolerans.
9. <partinfo>K525224</partinfo> - S-layer protein CspB with TAT-sequence: This fluorescent S-layer fusion protein is used to characterize the intracellular localisation and purification methods for CspB S-layers from Corynebacterium halotolerans.
10. <partinfo>K525304</partinfo> - Fusion protein of S-layer SbpA and mCherry: Characterization of induction and expression profiles of this S-layer fusion protein.
11. <partinfo>K525306</partinfo> - Fusion protein of S-layer SgsE and mCerulean: Characterization of induction and expression profiles of this S-layer fusion protein.
12. <partinfo>K525406</partinfo> - Fusion protein of S-layer SbpA and mCerulean: Characterization of induction and expression profiles of this S-layer fusion protein.
13. <partinfo>K525311</partinfo> - Fusion protein of S-layer SgsE and firefly luciferase: Characterization of induction and expression profiles, purification and stability of luciferase activity of this S-layer fusion protein.
14. <partinfo>K525551</partinfo> - Polycistronic expression of FNR, BisdA and BisdB: Characterization of the BPA degradation ability of this device.
15. <partinfo>K525562</partinfo> - Expression of fusion protein of FNR, BisdA and BisdB: Characterization of the BPA degradation ability of this device.
16. <partinfo>K525582</partinfo> - Polycistronic expression of FNR and fusion protein BisdA and BisdB: Characterization of the BPA degradation ability of this device.