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Project Proposal for iGEM 2007 - Project Specifications -

Detect pH of a system. Maintain a neutral pH in the system. Project Proposal for iGEM 2007 - Project Specifications -. Genetically Engineered pH Buffering Machine. Report pH of a system. Standard buffering reagents get used up. Breaking ground:.

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Project Proposal for iGEM 2007 - Project Specifications -

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  1. Detect pH of a system • Maintain a neutral pH in the system Project Proposal for iGEM 2007- Project Specifications - Genetically Engineered pH Buffering Machine • Report pH of a system

  2. Standard buffering reagents get used up Breaking ground: • No well-characterized pH-inducible/ pH-regulatory parts in • the BioBrick registry to date Project Proposal for iGEM 2007- Project Specifications - • pH is an important parameter Why is the project worth pursuing?

  3. Project Proposal for iGEM 2007- Background Information- Kinetics of expression of the E.coli cad operon as a function of pH and lysine. Melody N. Neely and Eric R. Olson. Journal of Bacteriology. Vol. 178, pp5522-28 (1996)

  4. Project Proposal for iGEM 2007- Background Information- Characterization of a pH-inducible promoter system for high-level expression of recombinant proteins in E.coli. Chou et al. Biotech and Bioeng. Vol. 47, pp186-192 (1995)

  5. IPTG Low pH LacI RFP CadC GFP Pconst PLac PCad CadC LacI RFP GFP Cell growth Total Pcad induction Specific Pcad induction pH 6.0 6.5 7.0 7.5 8.0 Project Proposal for iGEM 2007- Project Plan - (stage 1) E.coli cell

  6. IPTG Low pH LacI RFP CadC HrpR GFP Pconst PLac PCad LacI CadC HrpS Urease HrpR RFP HrpS HrpR HrpS Urease PAra PHrpL AraC Project Proposal for iGEM 2007- Project Plan - (stage 2) E.coli cell

  7. Urease + Urea Carbon dioxide Ammonia Project Proposal for iGEM 2007- Project Plan - (stage 2) pH increases √

  8. TetR CadC LacI HrpR TetR HrpR HrpS RFP Urease HrpS Lactate dehydrogenase HrpS Urease PAra PHrpL PTet Lactate dehydrogenase AraC IPTG Project Proposal for iGEM 2007- Project Plan - (stage 3) Low pH LacI RFP CadC HrpR GFP Pconst PLac PCad E.coli cell

  9. Pyruvate Lactic acid Lactate dehydrogenase Project Proposal for iGEM 2007- Project Plan - (stage 3) pH decreases √

  10. Project Proposal for iGEM 2007- Timeline - • Modelling (Week 3 – 5) • - Model expression and interactions of proteins involved • - Send DNA sequences for synthesis • Implementation (Week 5 – 9) • - Calibrate pH dependence of Pcad induction • - Relate green/ red fluorescence to pH • - Express urease and lactate dehydrogenase in E.coli • - Verify if enzymes change pH of systems as expected • Testing/ Validation (Week 9 – 10) • - Combine different components • - Test system at a range of pH

  11. Project Proposal for iGEM 2007- Possible Challenges - • Calibration of pH vs. fluorescence • Expression of urease and lactate dehydrogenase • - Use alternative pH-changing enzymes • - e.g. amino acid decarboxylases/ deaminases

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