IGEM:Harvard/2006/Cyanobacteria

"Required Reading"

Cyanobacteria backround and practicality

1. Genetic noise in the cyanobacterial oscillator
   • Very good PhD thesis by Jeffrey Chabot which has general cyanobacteria information and culturing information. Also deals with using a GFP reporter. From the vanO lab.
2. Working with algae
   • General information on growing cyanobacteria
3. Light conversion guide
   • Conversion information of units supplied by Peter Weigele.

Papers on latest findings

1. Nakajima M, Imai K, Ito H, Nishiwaki T, Murayama Y, Iwasaki H, Oyama T, and Kondo T. Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro. Science. 2005 Apr 15;308(5720):414-5. DOI:10.1126/science.1108451 |
2. Kondo T and Ishiura M. The circadian clock of cyanobacteria. Bioessays. 2000 Jan;22(1):10-5. DOI:10.1002/(SICI)1521-1878(200001)22:1<10::AID-BIES4>3.0.CO;2-A |
3. Naef F. Circadian clocks go in vitro: purely post-translational oscillators in cyanobacteria. Mol Syst Biol. 2005;1:2005.0019. DOI:10.1038/msb4100027 |
4. Xu Y, Mori T, and Johnson CH. Cyanobacterial circadian clockwork: roles of KaiA, KaiB and the kaiBC promoter in regulating KaiC. EMBO J. 2003 May 1;22(9):2117-26. DOI:10.1093/emboj/cdg168 |

1. Implementing KaiABC in-vitro and demonstrating circadian oscillation
   • Hetmann's review here
2. Good review paper on cyanobacteria oscillator, featuring clearly what is unknown
   • Peng's review here
3. General review paper of current cyanobacteria knowledge recommended by Dave
4. Information on measuring Phosphorylated KaiC
   • See here for review from paper + image

Incubator/Supplies

• Incubator Dimensions: 20 x 20 in.
• Lighting: 500 foot candles of cool white fluorescent
  • A 40-watt bulb at about 15cm will provide 500 foot candles of illumination. This will fall to about 200 foot candles at 50cm.
  • Need a light meter to measure illumination.
• Shopping List:
  • 2-3 20 x __ cool white fluorescent light fixtures. 40w?
  • 20x20 plexiglass sheet
  • wire foil
  • duct tape
  • Timer (hour increments)
  • extension cord compatible with timer
  • Some way to move the brackets/shelf in the incubator

Possible Molecular Mechanisms

After a review of literature, one will find that not much is known about the molecular mechanism; we only have theories of what could be true. Below are some of them, evidance that supports it, and evidance to the contrary.

Activator/Repressor

• Barkai and Leibler 2000
• Modeled after Eukarayotic systems
• Probably not true
  • KaiABC vary as activator/repressor
  • Transcription/translation not essential (invitro experiment)

"Activator-repressor model from 2000, by Chabot 2005"

KaiC phosphorylation model

• Xu et al 2003
• Previous research showed
  • Cells without KaiA had all unphosphorylated KaiC
  • Cells without KaiB has all phosphorylated KaiC
  • KaiA protein constant
  • Iwasaki et al. 2002
• Note: If this model holds true than our experiment in E. coli should show some silencing of genes downstream of KaiBC? We could test it by putting a reporter right downstream of KaiBC easily... very interesting.
• Note: This would be a good question to ask someone: if we put a reporter right downstream of KaiBC what should happen to that reporter.

"KaiC phosphorylation model from 2003, by Chabot 2005"

KaiB spaitotemporal localization model

• Kitayama et al 2003
• Idea that KaiB rotates location from the membrane to cytosol
  • Doesn't the in-vitro experiment disprove this?

"KaiB spaitotemporal localization, 2003"

Transcriptional/Translational independent model

• Tomita, Nakajima et al 2005
• Minimal oscillator and an extended timing system
• Best oscillation system currently developed

"Transcriptional/translational independent, 2005"

KaiC helicase model

• Proposed by C Johnson (unpub.)
• Looked at the 2 endogenous plasmids in cyanobacteria and found that they varied supercoiling state
• Hypothesis is that KaiC acts as a helicase which controls transcription access over genes

Important People

1. Prof. Alexander van Oudenaarden MIT
   • Emailed, we can meet up with him when he gets back from Woods Hole first week of July
   • May have many of the genes we want already on plasmids
2. Jeffrey Chabot
   • Post Doc who wrote the PhD thesis on cyanobacteria
   • Probably knows a lot of information but I can't find his contact
3. Peter Weigele MIT
   • Post Doc who works with cyanobacteria
   • Have emailed corresondance (see Peng); can get PCC7942 strains
4. Prof. Andrew Knoll Harvard OEB
   • Worked with evolutionary cyanobacteria
   • Called; gave references to other experts around the area

Questions that we need to ask

1. If we put a reporter downstream of kaiBC what happens to it
2. What happens when we put a plasmid in cyanobacteria? Does it replicate?
   • There are endogenous plasmids (2)
   • PCC7942 has homologous recombination
     • But we don't know if a plasmid will be maintained
3. Do we know what the sigma factor is in Kondo et al
4. Possible ways for reporting if oscillation works
5. Any other mechanisms?
6. How bad is the codon bias problem / would we need to actually mutate parts of the genome to move to E. coli?
7. Is there any feasible reporter we can have in E. coli?

Project Presentation

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Scratchpad

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