Transcript A synthetic gene-metabolic oscillator

A synthetic genemetabolic oscillator
Fung et al. 2005
Conceptual
design of
oscillatory
circuit
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.
E. coli 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.
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.
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.
ODE model
Metabolites (AcCoA, AcP, OAc−, and
HOAc)
Proteins LacI, Pta,
and Acs
Enzyme kinetics
Michaelis-Menten kinetics
V  Vmax
[S ]
KM  [S ]
K M is the Michaelis-Menten constant.
http://en.wikipedia.org/wiki/Michaelis-Menten
Gene expression kinetics
Hill function
TF binding reaction:
nTF  P  TFP
At equilibrium,
[TF ]n [ P]  K n [TFP] where K describes the binding
affinity between the TF and promoter.
The fraction of promoter binding sites is
[TFP]
[TF ]n
 

[ P]  [TFP]
K n  [TF ]n
The expression rate is
Ractivation      
  ([TF ]/ K )n
1  ([TF ]/ K )
Rrepression    (1   )   
n


1  ([TF ]/ K )
n

Rate expressions
and parameters
Michaelis-Menten
kinetics
Gene expression
kinetics
Simulations
Simulations
Simulations
Experimental verification
Fructose
Glycolytic flux decreases in the following
order: glucose, fructose, mannose, and
glycerol.
mannose
glycerol
Stability analysis
i is related to the gene copy number
for the synthesis of protein i (where i
is LacI, Pta or Acs).
Experimental result
Effect of acetate on metabolator dynamics. 10mM external
acetate suppressed oscillation but 0.1mM did not.
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.