Harvard iGEM 2006 Cyanobacterial Oscillator From cyanobacteria......to E. coli Photosynthetic Circadian rhythm Evolved over billions of years Model organism.

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