Project Proposal for iGEM 2007 - Project Specifications -
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Transcript of Project Proposal for iGEM 2007 - Project Specifications -
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
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?
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)
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)
Project Proposal for iGEM 2007
- Project Plan - (stage 1)
pH6.0 8.06.5 7.0 7.5
Cell growth
Total Pcad induction
Specific Pcad induction
E.coli cell
Pconst
LacI
LacI
GFPCadC
PLac
IPTG
PCad
Low pH
CadC
GFP
RFP
RFP
IPTG
PLac
CadC
CadC
GFPPCad
RFP
RFP
Pconst
LacI
LacI Low pH
Project Proposal for iGEM 2007
- Project Plan - (stage 2)
E.coli cell
HrpR
HrpR
PAraHrpS
PHrpLUrease
Urease
HrpSHrpR
HrpS
AraC
Project Proposal for iGEM 2007
- Project Plan - (stage 2)
Urease
UreaCarbon dioxide
+ Ammonia
pH increases √
IPTG
PLac
CadC
CadC
GFPPCad
RFP
RFP
Pconst
LacI
LacI Low pH
Project Proposal for iGEM 2007
- Project Plan - (stage 3)
E.coli cell
HrpR
HrpR
PAraHrpS
PHrpLUrease
Urease
HrpSHrpR
HrpS
AraC
TetR
TetR
Lactate dehydrogenase
Lactate dehydrogenase
PTet
Project Proposal for iGEM 2007
- Project Plan - (stage 3)
Lactate dehydrogenase
PyruvateLactic acid
pH decreases √
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
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