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Harvard iGEM 2006
Cyanobacterial Oscillator
From cyanobacteria...
...to E. coli
• Photosynthetic
• Circadian rhythm
• Evolved over billions of years
• Model organism for synthetic biology
• BioBrick registry
Harvard iGEM 2006
Cyanobacterial Oscillator
Time
Applications of a Bio-oscillator
hu
-Clock-Nightlight-Timed drug delivery-Bio-circuitry-Scientific research
Harvard iGEM 2006
Cyanobacterial Oscillator
The Repressilator Our Bio-oscillator
LacLac
Λ-cIΛ-cI
TetTet
Time
Fluo
resc
ence
Elowitz et al. 2000
Harvard iGEM 2006
Cyanobacterial Oscillator
P P
P
P
P
P
The Kai Clock in Cyanobacteria
•KaiC autophosphorylates and dephosphorylates•KaiA promotes phosphorylation•KaiB inhibits KaiA
Time
Phos
phor
ylati
on o
f Kai
C
Harvard iGEM 2006
Cyanobacterial Oscillator
Project GoalsReconstitute the cyanobacteria Kai oscillator in E. coli
1. Create KaiA, KaiB, and KaiC BioBricks.
2. Combine the above with promoters, etc. to form functional BioBricks.
3. Express Kai proteins in E. coli and verify interaction.
4. Verify oscillation in E. coli.
Harvard iGEM 2006
Cyanobacterial Oscillator
Results: Construct Creation
We’ve made the following constructs:
Kai genes
Lac promoter + Kai genes
KaiA + KaiC and KaiB + KaiC (with promoters)
Harvard iGEM 2006
Cyanobacterial Oscillator
P P
P
P
P
P
Results: Protein Interaction
Harvard iGEM 2006
Cyanobacterial Oscillator
Results: Protein Interaction
• Constructs were transformed in E. coli• Cultures were grown overnight• Western blot was washed with anti-KaiC antibodies (courtesy
of Susan Golden)• The gel was imaged, producing the above results containing
phosphorylated and unphosphorylated KaiC
Western blot Image
Anti-KaiC antibodies
Harvard iGEM 2006
Cyanobacterial Oscillator
Results: Protein InteractionWestern blot results
Anti-KaiC antibodies
P P
P
P
P
P
Harvard iGEM 2006
Cyanobacterial Oscillator
Results: Protein InteractionWestern blot results (interaction experiment)
Anti-KaiC antibodies
P P
P
P
P
P
Harvard iGEM 2006
Cyanobacterial Oscillator
Results: Protein InteractionWestern blot results (interaction experiment)
Anti-KaiC antibodies
P P
P
P
P
P
Harvard iGEM 2006
Cyanobacterial Oscillator
Results: Protein InteractionWestern blot results (interaction experiment)
Anti-KaiC antibodies
P P
P
P
P
P
Harvard iGEM 2006
Cyanobacterial Oscillator
Acknowledgements• Our teaching fellows 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 Texas A&M for providing anti-KaiC antibodies and advice
• Peter Wiegele @ MIT and Eric Webb @ WHOI for cyanobacteria strains