A synthetic gene-A synthetic gene-metabolic oscillatormetabolic oscillator

Fung et al. 2005Fung et al. 2005

ConceptuConceptual design al design

of of oscillatory oscillatory

circuitcircuit

Realization of the oscillatory circuit using the Realization of the oscillatory circuit using the acetate pathway in E. acetate pathway in E. colicoli

AcP: acetyl phosphateOAc-: acetateHOAc protonated form of acetate

Pta: phosphate acetyltransferaseAck: acetate kinaseAcs: acetyl-CoA synthetase

The induction of Acs by the presence of acetate is under catabolite repression by glucose in wild-type strain so as to avoid futile cycling.

E. E. colicoli strain and plasmid constructs strain and plasmid constructs

AcP

Acs LacI

Pta

Acetyl phosphate in the M2 pool activates acs expression, and at the same time, represses pta expression indirectly.

A GFP is ligated downstream of a plasmid-borne LacI-repressible tac promoter, to be used as a readout.

RepresentativRepresentative single cell e single cell dynamicsdynamics

About 60% of the initial colonies oscillate with varying amplitude and a period of 45 ± 10 min (for a total of 85 cells in 10 experiments), for a total duration of at least 4 h.

Representative oscillation dynamics of two Representative oscillation dynamics of two sibling cellssibling cells

Comparison of sibling cells shows skipped or delayed peaks (compare filled and open circles), which might be partially explained by uneven distribution of cellular materials.

ODE modelODE model

Metabolites (AcCoA, AcP, OAc−, and HOAc)

Proteins LacI, Pta, and Acs

Enzyme kineticsEnzyme kinetics

http://en.wikipedia.org/wiki/Michaelis-Menten

max

[ ]

[ ]M

SV V

K S

Michaelis-Menten kinetics

MK is the Michaelis-Menten constant.

Gene expression kineticsGene expression kineticsHill function

TF binding reaction:

[ ] [ ] [ ]n nTF P K TFP

nTF P TFP

[ ] [ ]

[ ] [ ] [ ]

n

n n

TFP TF

P TFP K TF

([ ] / )

1 ([ ] / )

n

activation n

TF KR

TF K

At equilibrium,

where K describes the binding affinity between the TF and promoter.

The fraction of promoter binding sites is

The expression rate is

(1 )1 ([ ] / )repression n

RTF K

Rate expressions and parameters

Michaelis-Menten kinetics

Gene expression kinetics

SimulationsSimulations

SimulatioSimulationsns

SimulatioSimulationsns

Experimental Experimental verificationverification

Fructose

mannose

glycerol

Glycolytic flux decreases in the following order: glucose, fructose, mannose, and glycerol.

Stability analysisStability analysis

is related to the gene copy number for the synthesis of protein i (where i is LacI, Pta or Acs).

i

Experimental resultExperimental result

Effect of acetate on metabolator dynamics. 10mM external acetate suppressed oscillation but 0.1mM did not.

DiscussionsDiscussions

► The unique design of the metabolator representThe unique design of the metabolator represents a fusion of metabolic and transcriptional oscills a fusion of metabolic and transcriptional oscillation.ation.

► The metabolator illustrates the use of metabolic The metabolator illustrates the use of metabolic fluxes to control biological oscillation.fluxes to control biological oscillation.

► Negative feedback is typically used to stabilize a Negative feedback is typically used to stabilize a system, however, it tends to be unstable if the tisystem, however, it tends to be unstable if the time lag in feedback increases.me lag in feedback increases.