Team:Bielefeld-Germany/Data Page
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- | This page gives a basic overview about our cell-free | + | 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 [[Team:Bielefeld-Germany/Project/Description | project description]] as well as in the [[Team:Bielefeld-Germany/Project/Background/BPA | bisphenol A]], [[Team:Bielefeld-Germany/Project/Background/S-Layer | S-layer]] and [[Team:Bielefeld-Germany/Project/Background/NAD | NAD<sup>+</sup> detection]] background subsections. |
- | ==How our | + | ==How our system works== |
- | [[Image:Bielefeld_2011_S-Layer-Produktion_lysis-purification_v1.jpg|center|700px|thumb|'''Figure 1: Production of the S- | + | [[Image:Bielefeld_2011_S-Layer-Produktion_lysis-purification_v1.jpg|center|700px|thumb|'''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).]] |
- | [[Image:Bielefeld_2011_Bead-Coating_v1.jpg|center|700px|thumb|'''Figure 2: Coating of the silica beads with the S- | + | [[Image:Bielefeld_2011_Bead-Coating_v1.jpg|center|700px|thumb|'''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.]] |
+ | [[Image:IGEM_Bielefeld_Project.jpg|center|700px|thumb|'''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<sup>+</sup> 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<sup>+</sup>-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== | |
+ | # <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. | ||
+ | # <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. | ||
+ | # <partinfo>K525710</partinfo> - '''NAD<sup>+</sup>-dependent DNA ligase from ''E. coli'' (LigA) ''': This enzyme enables determination of NAD<sup>+</sup> even in very low concentrations by introducing it to a molecular beacon based bioassay. | ||
- | ==Data | + | ==Data for pre-existing parts== |
- | # [http://partsregistry.org/Part: | + | # [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<sup>-1</sup> Bisphenol A with polycistronic ''bisdAB'' gene in 30 - 33 h. Even faster (21 - 24 h) when using a fusion protein of BisdA and BisdB. |
- | + | # [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<sup>-1</sup> Bisphenol A with polycistronic ''bisdAB'' gene in 30 - 33 h. Even faster (21 - 24 h) when using a fusion protein of BisdA and BisdB. | |
- | # [http://partsregistry.org/Part: | + | |
- | == | + | ==We have also characterized the following parts== |
- | # | + | # <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. |
- | + | # <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>. | |
- | + | # <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. | |
- | + | # <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''. | |
- | + | # <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''. | |
- | + | # <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''. | |
- | + | # <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''. | |
- | # | + | # <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''. |
- | # | + | # <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''. |
- | # | + | # <partinfo>K525304</partinfo> - '''Fusion protein of S-layer SbpA and mCherry''': Characterization of induction and expression profiles of this S-layer fusion protein. |
+ | # <partinfo>K525306</partinfo> - '''Fusion protein of S-layer SgsE and mCerulean''': Characterization of induction and expression profiles of this S-layer fusion protein. | ||
+ | # <partinfo>K525406</partinfo> - '''Fusion protein of S-layer SbpA and mCerulean''': Characterization of induction and expression profiles of this S-layer fusion protein. | ||
+ | # <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. | ||
+ | # <partinfo>K525551</partinfo> - '''Polycistronic expression of FNR, BisdA and BisdB''': Characterization of the BPA degradation ability of this device. | ||
+ | # <partinfo>K525562</partinfo> - '''Expression of fusion protein of FNR, BisdA and BisdB''': Characterization of the BPA degradation ability of this device. | ||
+ | # <partinfo>K525582</partinfo> - '''Polycistronic expression of FNR and fusion protein BisdA and BisdB''': Characterization of the BPA degradation ability of this device. |
Latest revision as of 20:54, 28 October 2011
![](https://static.igem.org/mediawiki/2011/3/3c/Bielefeld-Germany2011-header-full.png)
![](https://static.igem.org/mediawiki/2011/f/ff/Bielefeld-header-data-page.png)
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
![](/wiki/images/d/d7/IGEM_Bielefeld_Project.jpg)
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
- <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.
- <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.
- <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
- [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.
- [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
- <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.
- <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>.
- <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.
- <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.
- <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.
- <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.
- <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.
- <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.
- <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.
- <partinfo>K525304</partinfo> - Fusion protein of S-layer SbpA and mCherry: Characterization of induction and expression profiles of this S-layer fusion protein.
- <partinfo>K525306</partinfo> - Fusion protein of S-layer SgsE and mCerulean: Characterization of induction and expression profiles of this S-layer fusion protein.
- <partinfo>K525406</partinfo> - Fusion protein of S-layer SbpA and mCerulean: Characterization of induction and expression profiles of this S-layer fusion protein.
- <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.
- <partinfo>K525551</partinfo> - Polycistronic expression of FNR, BisdA and BisdB: Characterization of the BPA degradation ability of this device.
- <partinfo>K525562</partinfo> - Expression of fusion protein of FNR, BisdA and BisdB: Characterization of the BPA degradation ability of this device.
- <partinfo>K525582</partinfo> - Polycistronic expression of FNR and fusion protein BisdA and BisdB: Characterization of the BPA degradation ability of this device.