IGEM:Harvard/2006/Cyanobacteria: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 90: Line 90:
*Nakajima et. al: ''in vitro'', the KaiABC proteins oscillate robustly by themselves, albeit with a lower amplitude than ''in vivo''
*Nakajima et. al: ''in vitro'', the KaiABC proteins oscillate robustly by themselves, albeit with a lower amplitude than ''in vivo''


'''Don't delete'''From Xu et al 2004, they have anti-KaiC polyclonal antibodies which they used to do a phosphorylated KaiC assay; we can use this method to analyse KaiC fluctuation.
'''Don't delete'''
 
From the paper:
  KaiC Phosphorylation Assay. Cyanobacterial cultures were grown to
  an OD750 of 0.2. After a 12-h dark pulse, the cultures were treated
  with 10�MIPTGfor 3 h at 30°C in light with air bubbling, and�40
  ml of the cultures were collected for preparation of total extracts.
  Immunoblot analysis for KaiC was performed on 5 �g of total
  proteins per lane according to the previous description (20), except
  that we used a highly specific mouse polyclonal antibody to KaiC
  and a SuperSignalWest Pico Chemiluminescent Substrate (Pierce).
  The phosphorylated KaiC signals were quantified as before (16)
  and analyzed by Student’s t test.
 
[[Image: Xu_gelp.jpg |thumb| "Image of figure shown in Xu paper"]]

Revision as of 09:08, 21 June 2006

"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 | PubMed ID:15831759 | HubMed [1]
  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 | PubMed ID:10649285 | HubMed [2]
  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 | PubMed ID:16729054 | HubMed [3]
  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 | PubMed ID:12727878 | HubMed [4]

All Medline abstracts: PubMed | HubMed

  1. Implementing KaiABC in-vitro and demonstrating circadian oscillation
  2. Good review paper on cyanobacteria oscillator, featuring clearly what is unknown
  3. General review paper of current cyanobacteria knowledge recommended by Dave
  4. Information on measuring Phosphorylated KaiC

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

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"

Possible Project Ideas

People to contact

Questions that we need to ask

  • Q: If we put a reporter downstream of kaiBC what happens to it
  • Q: Do we know what the sigma factor is in Kondo et al
  • Q: Possible ways for reporting if oscillation works
  • Q: Any other mechanisms?
  • Q: How bad is the codon bias problem / would we need to actually mutate parts of the genome to move to E. coli?

Project Presentation

Moved to new page

3000bp, 11cents/base. --> synthetic


  • Nakajima et. al: in vitro, the KaiABC proteins oscillate robustly by themselves, albeit with a lower amplitude than in vivo

Don't delete

Retrieved from "https://openwetware.org/mediawiki/index.php?title=IGEM:Harvard/2006/Cyanobacteria&oldid=43732"