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