Harvard Cyano-Oscillator

1
Harvard Cyano-Oscillator

• Hetmann Hsieh
• Jeffrey Lau
• David Ramos
• Zhipeng Sun

2
Outline

• Introduction
• Motivation
• Kai Protein Interactions
• Project Goals
• Obtaining the Kai Proteins
• Biobricking the Kai Proteins
• Stage I Expression and Interaction of Kai
  Proteins in E. coli
• Stage II Synchronization of Kai Proteins in E.
  coli
• Stage III Future Possibilities
• Conclusion

3
Motivation

• The repressilator
• Transcriptional repression system
• Plasmid to right, GFP reporter
• Lite means destruction tag
• T200min
• Is not stable over time
• Problem with implementation in later generations
  of the repressillator

• Why a cyanobacterial oscillator instead?
• Rhythm has been shown to be driven by the
  interaction of three proteins, KaiABC, which are
  sufficient to produce oscillation in vitro
  without transcription regulation (Nakajima et
  al., 2005).
• Cyanobacteria oscillation is robust, stable over
  time, and temperature-independent (within living
  tolerances).
• The oscillation period can be adjusted from
  14-60hours by point mutations of KaiC (Kondo et
  al., 2000).
• Post translational mechanism means less energy
  required. (keep?)

Elowitz et al. 2000
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Project Goals

• Reconstitute the cyanobacteria KaiABC oscillator
  in E. coli

1. Create KaiA, KaiB, and KaiC Biobricks.
2. Transform E. coli with Kai Biobricks to
   reconstitute KaiC phosphorylation cycle (no
   reporter attached).
3. Distant Transform E. coli with Kai Briobricks to
   reconstitute KaiC phosphorylation cycle with
   Biobricked reporter.

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Obtaining the Kai Proteins

• Extracting from PCC7942 cyanobacteria

6
Obtaining the Kai Proteins II

• DNA synthesis

Synthesized KaiA, KaiB, and KaiC separately, with
biobrick prefixes and suffixes and extra stop
codons. Also included two point mutations on
KaiC to remove the PstI and EcoRI sites to ensure
biobricks compatibility. Ordered from Geneart
and received correct sequences in 18 days.