Download Project Proposal for iGEM 2007

Project Proposal for iGEM 2007
- Project Specifications -
Genetically Engineered
pH Buffering Machine
• Detect pH of a system
• Report pH of a system
• Maintain a neutral pH in the system
Project Proposal for iGEM 2007
- Project Specifications Why is the project worth pursuing?
• pH is an important parameter
• 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
- Background InformationKinetics 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 InformationCharacterization 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)
IPTG
CadC
Low pH
LacI
RFP
GFP
LacI
Pconst
CadC
PLac
RFP
GFP
PCad
E.coli cell
Cell growth
Total Pcad induction
Specific Pcad induction
6.0
6.5
pH
7.0
7.5
8.0
Project Proposal for iGEM 2007
- ProjectIPTG
Plan - (stage 2)
CadC
Low pH
LacI
LacI
Pconst
CadC
RFP
GFP
PLac
HrpR
PCad
HrpR
HrpS
HrpR
HrpS
HrpS
PAra
RFP
Urease
PHrpL
Urease
AraC
E.coli cell
Project Proposal for iGEM 2007
- Project Plan - (stage 2)
Urease
Urea
Carbon
dioxide
+
Ammonia
pH increases √
Project Proposal for iGEM 2007
- Project
Plan
(stage
3)
IPTG
CadC
Low pH
LacI
LacI
Pconst
CadC
RFP
GFP
PLac
TetR
HrpR
TetR
HrpS
HrpS
HrpS
Lactate dehydrogenase
Urease
PHrpL
PTet
Urease
AraC
HrpR
PCad
HrpR
PAra
RFP
Lactate
dehydrogenase
E.coli cell
Project Proposal for iGEM 2007
- Project Plan - (stage 3)
Lactate
dehydrogenase
Pyruvate
Lactic
acid
pH decreases √
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