A synthetic genemetabolic oscillator

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A synthetic gene-metabolic oscillator

• Fung et al. 2005

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Conceptual design of oscillatory circuit
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Realization of the oscillatory circuit using the
acetate pathway in E. coli
AcP acetyl phosphate OAc- acetate HOAc
protonated form of acetate
Pta phosphate acetyltransferase Ack acetate
kinase Acs 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.
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E. coli strain and plasmid constructs
A GFP is ligated downstream of a plasmid-borne
LacI-repressible tac promoter, to be used as a
readout.
Acetyl phosphate in the M2 pool activates acs
expression, and at the same time, represses pta
expression indirectly.
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Representative single cell dynamics
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.
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Representative oscillation dynamics of two
sibling 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.
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ODE model
Metabolites (AcCoA, AcP, OAc-, and HOAc)
Proteins LacI, Pta, and Acs
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Enzyme kinetics
Michaelis-Menten kinetics
is the Michaelis-Menten constant.
http//en.wikipedia.org/wiki/Michaelis-Menten
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Gene expression kinetics
Hill function
TF binding reaction
At equilibrium,
where K describes the binding affinity between
the TF and promoter.
The fraction of promoter binding sites is
The expression rate is
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Rate expressions and parameters
Michaelis-Menten kinetics
Gene expression kinetics
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Simulations
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Simulations
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Simulations
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Experimental verification
Fructose
Glycolytic flux decreases in the following order
glucose, fructose, mannose, and glycerol.
mannose
glycerol
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Stability analysis
is related to the gene copy number for the
synthesis of protein i (where i is LacI, Pta or
Acs).
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Experimental result
Effect of acetate on metabolator dynamics. 10mM
external acetate suppressed oscillation but 0.1mM
did not.
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Discussions

• The unique design of the metabolator represents a
  fusion of metabolic and transcriptional
  oscillation.
• The metabolator illustrates the use of metabolic
  fluxes to control biological oscillation.
• Negative feedback is typically used to stabilize
  a system, however, it tends to be unstable if the
  time lag in feedback increases.