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

2. Breaking ground: Project Proposal for iGEM 2007 - Project Specifications - pH is an important parameter No well-characterized pH-inducible/ pH-regulatory parts in the BioBrick registry to date Standard buffering reagents get used up 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. Project Proposal for iGEM 2007 - Project Plan - (stage 1) pH 6.08.06.57.07.5 Cell growth Total Pcad induction Specific Pcad induction E.coli cell Pconst LacI GFP CadC PLac IPTG PCad Low pH CadC GFP RFP

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

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

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

9. Project Proposal for iGEM 2007 - Project Plan - (stage 3) Lactate dehydrogenase Pyruvate Lactic acid 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 - Expression of urease and lactate dehydrogenase - Use alternative pH-changing enzymes - e.g. amino acid decarboxylases/ deaminases Calibration of pH vs. fluorescence