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.

Contents

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, BBa_K525499), ferredoxin (Fd, BBa_K123000) and cytochrome P450 (CYP, BBa_K123001) which are fused to a S-layer protein. The molecular beacon (hairpin structure) binds two short DNA oligos. The NAD+-dependent ligase (LigA, BBa_K525710), 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. BBa_K525305 - 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. BBa_K525515 - 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. BBa_K525710 - 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. BBa_K525405 - 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. BBa_K525512 - Polycistronic expression of BisdA and BisdB: This is the version of BPA degrading BioBricks found in the partsregistry - comparison to our fusion protein BBa_K525515.
  3. BBa_K525517 - 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. BBa_K525234 - 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. BBa_K525121 - 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. BBa_K525123 - 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. BBa_K525222 - 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. BBa_K525223 - 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. BBa_K525224 - 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. BBa_K525304 - Fusion protein of S-layer SbpA and mCherry: Characterization of induction and expression profiles of this S-layer fusion protein.
  11. BBa_K525306 - Fusion protein of S-layer SgsE and mCerulean: Characterization of induction and expression profiles of this S-layer fusion protein.
  12. BBa_K525406 - Fusion protein of S-layer SbpA and mCerulean: Characterization of induction and expression profiles of this S-layer fusion protein.
  13. BBa_K525311 - 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. BBa_K525551 - Polycistronic expression of FNR, BisdA and BisdB: Characterization of the BPA degradation ability of this device.
  15. BBa_K525562 - Expression of fusion protein of FNR, BisdA and BisdB: Characterization of the BPA degradation ability of this device.
  16. BBa_K525582 - Polycistronic expression of FNR and fusion protein BisdA and BisdB: Characterization of the BPA degradation ability of this device.