Transcript Cyanobacterial Oscillator Hetmann Hsieh Jeffrey Lau David Ramos Zhipeng Sun Harvard iGEM 2006 Background Project Goal Cyanobacteria, also known as blue-green algae, are a phylum of ancient photosynthetic bacteria.

Cyanobacterial Oscillator
Hetmann Hsieh
Jeffrey Lau
David Ramos
Zhipeng Sun
Harvard iGEM 2006
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:
1. Create KaiA, KaiB, and KaiC BioBricks.
2. Combine the above with registry parts to form functional BioBricks.
3. Express Kai proteins in E. coli and verify their interaction.
4. 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 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 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.
La
c
Nakajima et al.,
2005
P
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.
λcI
Te
t
Elowitz et al., 2000
P
P
P
P
P
Tomita et al., 2005
Construct Creation
Protein Interaction
Further challenges
We’ve created the following BioBricks
and added them to the registry:
Western blot shows expression and interaction of Kai
proteins.
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:
1
Kai genes
2
Kai genes +
Lac promoter
KaiA + KaiC
Lane 1: Lac + KaiC
Lane 2: Lac + KaiB + Lac + KaiC
Lane 3: Lac + KaiA + Lac + KaiC
3
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.
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, we’d expect to see no net oscillation
on our Western blot (see picture below).
Time
KaiB + KaiC
Results
References
We believe that the top band in each lane is phosphorylated KaiC while
the bottom band is non-phosphorylated KaiC.
M. B. Elowitz, S. Leibler, A synthetic oscillatory
network of transcriptional regulators. Nature 2000
Jan 20; 403(6767) 335-8.
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).
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.
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
KaiA+KaiB+KaiC construct, but the results from lane 2 are at least
consistent with our model.
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.