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The Zincolator ! Analytical tools for a S ynthetic Catalyst

The Zincolator ! Analytical tools for a S ynthetic Catalyst. Santiago Toledo & Robert Jonas Texas Lutheran University. Overall goals. Introduce students to the techniques and possibilities of synthetic biology. Develop a project to be incorporated in our sophomore level microbiology lab.

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The Zincolator ! Analytical tools for a S ynthetic Catalyst

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  1. The Zincolator!Analytical tools for a Synthetic Catalyst Santiago Toledo & Robert Jonas Texas Lutheran University

  2. Overall goals • Introduce students to the techniques and possibilities of synthetic biology. • Develop a project to be incorporated in our sophomore level microbiology lab. • Develop a student research project involving synthetic biology and current research ongoing in our departments. • Increase collaboration between the biology and chemistry departments.

  3. Biomimetic metal complex catalyst -Use a synthetic model of the active site containing Zn -Test the reactivity of the model complex towards substrate oxidation utilizing O2 as the single oxidant. Challenges: -Is the catalyst stable in water? -Is the catalyst utilizing O2 during the oxidation reaction? Szajna-Fuller, E. et al. Inorg. Chem. 2007, 46, 5499−5507

  4. Catalyst Stability • Is the Zn-containing catalyst stable in water? • We can design a sensor to detect the release of zinc • Assuming E. coli contains the ZntR/MerR zinc-sensing protein

  5. BioBricks-promoter • Part:BBa_K190022 • Designed by Michael Verhoeven   Group: iGEM09_Groningen   (2009-08-25) • Zinc Promoter (ZntR regulated) with own RBS • The pZntR from E.coli K.12 has a specific RBS site behind it in the genome. Here the RBS site is attached to the promoter region. The RBS site might influence the activity of the promoter and will be tested in the same way as BBa_K190016. ZntR activates transcription when Zn(II) is bound (1)

  6. BioBrick-reporter • Part:BBa_J18932 • Designed by Raik Gruenberg   Group: Affiliates   (2010-01-26) • From partsregistry.org • mCherry RFP • Red fluorescent protein derived from DsRed. • Advantages: • fast folding and maturation • bright and photo-stable • Degradation issues: • Ajo-Franklin...Silver (2007) report multiple bands for mCherry purifications in E. coli • SDS treatment or boiling can hydrolyze the chromophore position at F//MYG splitting the protein in half

  7. Schematic BBa_K190022 BBa_J18932 Promoter Reporter

  8. Oxygen Sensor • Since the reaction presumably uses oxygen, we can trace the progress of the reaction by tracking the depletion of oxygen • Assuming the E. coli has the Fnr oxygen sensor • We will have to control for metabolic use of oxygen by E. coli (running similar tubes with and without catalyst).

  9. BioBricks-Oxygen Sensor • Part:BBa_K239002 • Designed by Axel Nystrom   Group: iGEM09_UCL_London   (2009-06-22) • Currently in the planning stage. We could build it (160 bp). • HypB promoter, activated by Fnr during oxygen deficiency • Sequence contains 2 Fnr binding sites and is sigma70 dependent. Function: Switched on during anaerobic conditions (ca 7-fold). Area of application: Detection of when the cell is switching to anaerobic conditions.

  10. BioBrick-Reporter (same one) • Part:BBa_J18932 • Designed by Raik Gruenberg   Group: Affiliates   (2010-01-26) • From partsregistry.org • mCherry RFP • Red fluorescent protein derived from DsRed. • Advantages: • fast folding and maturation • bright and photo-stable • Degradation issues: • Ajo-Franklin...Silver (2007) report multiple bands for mCherry purifications in E. coli • SDS treatment or boiling can hydrolyze the chromophore position at F//MYG splitting the protein in half

  11. Schematic BBa_K239002 BBa_J18932 Promoter Reporter

  12. Thanks, Y’all! • We appreciate your help with this introduction to synthetic biology!

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