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A synthetic gene-A synthetic gene-metabolic oscillatormetabolic oscillator
Fung et al. 2005Fung et al. 2005
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.
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
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.