Cyanobacterial Oscillator Harvard iGEM 2006

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Cyanobacterial OscillatorHarvard iGEM 2006
Hetmann Hsieh Jeffrey Lau David Ramos Zhipeng Sun
Background
Project Goal
Cyanobacteria, also known as blue-green algae,
are a phylum of ancient photosynthetic bacteria.
They are the simplest organisms known to possess
a circadian rhythm. This rhythm is driven by the
interaction of three proteins, called KaiA, KaiB,
and KaiC. Our goal this summer was to
reconstitute the Kai oscillator in E. coli.

• To reconstitute the cyanobacterial Kai oscillator
  in E. coli
• We have achieved the first three of the following
  subgoals
• Create KaiA, KaiB, and KaiC BioBricks.
• Combine the above with registry parts to form
  functional BioBricks.
• Express Kai proteins in E. coli and verify their
  interaction.
• Verify oscillation in E. coli.

• Why care about biological oscillators in the
  first place? Bio-oscillators have a number of
  potential applications
• Regulating periodic tasks like drug delivery and
  pharmaceutical processes
• Synchronizing biological circuits.
• Aiding research into naturally-occurring
  oscillators

A flask of delicious green cyanobacteria sits on
our lab bench.
A Side-by-Side Comparison Repressilator and
Cyanobacteria
The repressilator, designed by Elowitz and
Leibler, is a famous synthetic biological
oscillator. It is driven by a triangle of mutual
transcriptional repression (see below) The
repressilator was a major achievement, but its
oscillation was not very stable. As shown in the
graph below, the trough of oscillation shifts
upward over time while the period increases.

• The cyanobacteria oscillator is driven by three
  proteins, named KaiA, KaiB, and KaiC. They are
  hypothesized to interact in the following way
  (see diagram at near right)
• KaiC exhibits spontaneous autokinase and
  autophosphorylase activity.
• KaiA promotes KaiC phosphorylation and inhibits
  KaiC dephosphorylation.
• KaiB inhibits KaiA effect on KaiC.
• The three Kai proteins are necessary and
  sufficient to create a stable oscillation in the
  phosphorylation state of KaiC.
• The cyanobacteria oscillator benefits from
  millions of years of evolution. It has a number
  of desirable features
• Stability over time
• Temperature compensation
• Period adjustable via point mutations in KaiC
  (Ishiura et al., 1998)
• Transcription-translation independence (
  minimal work to reconstitute the system in E.
  coli)
• The graph on the far right demonstrates stable
  oscillation of the Kai clock in vitro (Nakajima
  et al., 2005). These in vitro results give us
  confidence that the oscillator will work well in
  E. coli.

Nakajima et al., 2005
Elowitz et al., 2000
Tomita et al., 2005
Construct Creation
Protein Interaction
Further challenges
Western blot shows expression and interaction of
Kai proteins.
Weve created the following BioBricks and added
them to the registry
Our next step is to verify that the Kai clock is
actually oscillating over time in E. coli This
goal is completed by two problems of
synchronization Synchronization between cells
We need a way to synchronize the phases between
cells in a culture. If the cells in a culture are
out of phase, wed expect to see no net
oscillation on our Western blot (see picture
below).
Procedure We transformed three of our constructs
into E. coli, induced expression of the Kai
genes, and sampled the cultures at 0.55OD. We
lysed the samples and performed a Western blot
with anti-KaiC antibodies.
1 2 3
Kai genes
Kai genes Lac promoter
Lane 1 Lac KaiC Lane 2 Lac KaiB Lac
KaiC Lane 3 Lac KaiA Lac KaiC
KaiA KaiC
KaiB KaiC
Results We believe that the top band in each
lane is phosphorylated KaiC while the bottom band
is non-phosphorylated KaiC. As our model
predicts, the level of phosphorylated KaiC is
higher when KaiA and KaiC are combined (lane 3)
than when KaiC alone is expressed (lane 1). KaiB
KaiC (lane 2) appears the same as KaiC alone
(lane 1), which is consistent with our model
(since KaiB has no direct effect on KaiA). Our
results verify that KaiA and KaiC are being
expressed and interacting in E. coli. We cannot
verify KaiB protein interaction until we build
our KaiAKaiBKaiC construct, but the results
from lane 2 are at least consistent with our
model.
References
M. B. Elowitz, S. Leibler, A synthetic
oscillatory network of transcriptional
regulators. Nature 2000 Jan 20 403(6767) 335-8.
M. Ishiura et al., Expression of a gene cluster
kaiABC as a circadian feedback process in
cyanobacteria. Science 1998 Sep 4 281(5382)
1519-23 M. Nakajima et al., Reconstitution of
circadian oscillation of cyanobacterial KaiC
phosphorylation in vitro. Science 2005 Apr 15
308(5720) 414-5. J. Tomita et al., No
transcription-translation feedback in circadian
rhythm of KaiC phosphorylation. Science 2005 Jan
14 307(5707) 251-4.
Synchronization within cells We also need to
synchronize the phases of KaiC proteins within a
cell. If KaiC is continuously produced, the newly
generated proteins will be out of phase with
existing proteins in the cell. We would then
expect the net oscillation to decay over time.
See the graph above for output from a computer
model simulating this effect.
Solution We plan to solve both of these problems
by pulsing the expression of the Kai genes. This
will ensure that all the cells in a culture
initiate expression at the same time
(between-cell synchronization), and ensure that
expression ceases after a short period of time
(within-cell synchronization). We hope to have
these experiments running soon.