Cyanobacterial Oscillator

1
Cyanobacterial Oscillator
Creating a robust biological oscillating biobrick.

• Hetmann Hsieh
• Jeff Lau
• Dave Ramos
• Zhipeng Sun
• Harvard iGEM 2006

Cyanobacterial Oscillator
Harvard iGEM 2006
2
Motivation

• Repressilator (Elowitz and Leibler, 2000)
• Synthetic oscillator
• Unstable over time
• Our cyanobacterial oscillator
• Stable over long periods of time
• Period flexible from 14-60h

Cyanobacterial Oscillator
Harvard iGEM 2006
3
The Kai clock in cyanobacteria
Three proteins-- KaiA, KaiB, and KaiC interact
to produce oscillation in the phosphorylation
state of KaiC.

• Reconstituted in vitro with KaiABC proteins
  (Nakajima et al., 2005)
• Oscillation does not require transcriptional
  regulation
• Oscillation robust and temperature-resistant
• Many processes are regulated by the Kai
  phosphorylation cycle in cyanobacteria (including
  transcription of KaiBC)

Tomita et al, 2005
Cyanobacterial Oscillator
Harvard iGEM 2006
4
Project Goals

• Reconstitute the cyanobacteria KaiABC oscillator
  in E. coli
• Create KaiA, KaiB, and KaiC Biobricks.
• Ligate KaiABC BioBricks with promoters, etc. to
  form functional constructs.
• Transform E. coli with Kai constructs to
  reconstitute KaiC phosphorylation cycle (no
  reporter attached).
• Distant Implement intermediate transcription
  factors so the Kai clock can do useful work in E.
  coli.

Cyanobacterial Oscillator
Harvard iGEM 2006
5
Obtaining KaiABC Extraction
EcoRI
XbaI
PstI
EcoRI
PstI
kaiBC
kaiA
5
3
5
3
SpeI
3
A 3.1kb template containing KaiA, B, and C from cyanobacteria A 3.1kb template containing KaiA, B, and C from cyanobacteria
Advantage Disadvantage
Can be made into a biobrick compatible with cyanobacteria as well as basis for E. coli version Need three point mutations Need to split into KaiA, B, and C for insertion into e. coli
Result Had problems with cloning postponed until later Result Had problems with cloning postponed until later
Cyanobacterial Oscillator
Harvard iGEM 2006
6
Obtaining KaiABC Synthesis
Three separate constructs for each of KaiA, KaiB, and KaiC with biobrick ends Synthesis from Geneart GMBH Three separate constructs for each of KaiA, KaiB, and KaiC with biobrick ends Synthesis from Geneart GMBH
Advantage Disadvantage
Relatively fast (14 business days) Cannot make a cyanobacteria-compatible biobrick
Result Went with synthetic genes Result Went with synthetic genes
Cyanobacterial Oscillator
Harvard iGEM 2006
7
Experiments Interaction of KaiABC
R0010
B0034

• R0010 lac promoter
• B0034 strong RBS
• KaiC never phosphorylates

• KaiA phosphorylates KaiC

KaiC phosphorylation

• KaiB dephosphorylates KaiC

Time
Cyanobacterial Oscillator
Harvard iGEM 2006
8
Experiments Oscillation of KaiABC
KaiC phosphorylation
R0010
B0034 B0010

• Pulse KaiA once and pulse KaiB KaiC once
• Oscillation should weaken as KaiC degrades

Time
KaiC phosphorylation

• Constant KaiA, pulse KaiB KaiC once

• If KaiA phosphorylation and KaiC degradation at
  equal rates, should oscillate

Cyanobacterial Oscillator
Harvard iGEM 2006
9
Results

• When we get the KaiC construct working this will
  be filled D

Cyanobacterial Oscillator
Harvard iGEM 2006
10
Acknowledgements

• Our TFs Chris Doucette, Shawn Douglas, and Nick
  Stroustrup for their valuable help in lab
• Profs. Alain Viel, George Church, Pam Silver,
  William Shih, Radhika Nagpal, and Jagesh Shah
• Prof. Susan Golden at TAMU for helping us with
  Western blot antibodies and general advice
• Peter Wiegele _at_ MIT and Eric Webb _at_ WHOI for
  PCC7942 and PCC6803 strains

Cyanobacterial Oscillator
Harvard iGEM 2006