1. Cyanobacterial Oscillator 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 Background 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. A Side-by-Side Comparison: Repressilator and Cyanobacteria Lac λ-cI Tet 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. Project Goal 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. Construct Creation Kai genes (from PCC 7942 strain) Lac promoter KaiA + KaiC Protein Interaction Western blot shows expression and interaction of Kai proteins. KaiB + KaiC 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. Lane 1: Lac + KaiC Lane 2: Lac + KaiB + Lac + KaiC Lane 3: Lac + KaiA + Lac + KaiC 1 2 3 We’ve created the following BioBricks and added them to the registry: 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 the KaiA+KaiB+KaiC construct, but the results from lane 2 are at least consistent with our model. A flask of delicious green cyanobacteria sits on our lab bench. Further challenges 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, we’d expect to see no net oscillation on our Western blot (see picture below). 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. 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. Nakajima et al., 2005 + Kai genes PP P P P P Tomita et al., 2005 Elowitz et al., 2000