IGEM:Harvard/2006/Cyanobacteria

Agenda

Long-term project outline

For Monday

• Figure out optimal growing conditions (complete)
• Regrow cyanobacteria colonies (complete)
• Draft email questions for Professor Golden (complete)

For Tuesday

• PCR crash course (complete)
• Picnic! (complete)
• Check first batch of colonies again (complete)
• Complete more research in preparation for Professor Golden (complete)
• Order primers (complete)

For Wednesday

• Research transformation protocols (mostly complete)
• Start creating presentation for Friday
• Talk to Professor Golden (complete)
• Grow additional liquid cyanobacteria cultures (new order of PCC 7942 is not here yet)
• Finalize goals (work in cyanobacteria or in e. coli)/ plan out the rest of summer (in progress)

For Thursday

• Research Sigma factors (on hold)
• Design and order our first set of primers (for entire KaiABC sequence)
• Research plasmids to ask Prof. Golden about
  • Also, don't forget to ask her about CO2 and aerating our cultures
• Make presentation for Friday
• Find out what equipment we would need for measuring bioluminescence

For Friday

• Present before Fourth of July weekend

Notebook

<calendar> name=/Notebook date=2006/07/01 view=threemonths format=%name/%year-%month-%day weekstart=14 </calendar> For each page, newer entries are at the top.

Reading

Cyanobacteria background 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. Molecular biology of circadian rhythms / edited by Amita Sehgal
   • Peng's taking a look at it now; in Biolabs
3. Cyanosite
   • Lots of information on cyanobacteria culture mediums and good links to other resources.

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
5. More on measuring phosphorylated KaiC (SDS-PAGE)
   • Cited in the Nakajima paper

Incubator

Construction

We grow our cyanobacteria in a modified incubator with a timer-controlled fluorescent light. The incubator has a shaking floor.

• Interior dimensions: 50 cm x 50 cm x 60 cm tall
• Lighting: 32 W and 22 W fluorescent lightbulbs (circular shape). Measured light intensity on the floor varies from 3800-4500 lux on our flask

Incubator exterior

Incubator interior

Incubator interior (closeup)

Current status

This section will be updated to reflect the current contents of the incubator.

• Our light cycle is currently set to constant lighting.
  • I put the timer on the windowsill above the electrical outlet

Click here for a map of the lower shelf
(2006-6-28)

Click here for a map of the upper shelf
(2006-6-26)

Cyanobacteria care

• Optimum conditions for growth of cyanobacteria on solid media
  • Contains comparisons of many different media for several strains, including PCC7942 and PCC6803.
• Working with algae
  • General information on growing cyanobacteria
• Light conversion guide
  • Conversion information of units supplied by Peter Weigele.

Marine (WH8102)

Formula for SN medium

SN agar

Freshwater (PCC7942 and PCC6803)

See protocols. See research in conditions for growth.

Protocols

Main article: IGEM:Harvard/2006/Protocols/Cyanobacteria

Possible Molecular Mechanisms

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

Activator/Repressor

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

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

KaiC phosphorylation model

"KaiC phosphorylation model from 2003, by Chabot 2005"

• 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.

KaiB spaitotemporal localization model

"KaiB spaitotemporal localization, 2003"

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

Transcriptional/Translational independent model

"Transcriptional/translational independent, 2005"

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

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 to contact (/stalk)

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
   • We have his email address
3. Prof. Susan Golden Texas A&M
   • Is working on pretty much the same stuff we are looking at
   • Have emailed
   • Have talked to on phone-- see here
4. Peter Weigele MIT
   • Post Doc who works with cyanobacteria
   • Have emailed correspondance (see Peng); can get PCC7942 strains
   • Have contacted and obtained PCC7942 and PCC6803 (a glucose-digesting strain)
5. Prof. Andrew Knoll Harvard OEB
   • Worked with evolutionary cyanobacteria
   • Called; gave references to other experts around the area
6. Prof. Stjepko Golubic BU
   • Prof. Knoll said he knew a lot about cyanobacteria
7. Prof. Colleen Cavanaugh Harvard OEB
8. Prof. Woodland Hastings Harvard OEB
   • Corresponded with a Japanese lab group which works with cyanobacteria
   • Specializes in circadian rhythms

Questions that we need to ask

1. If we put a reporter downstream of kaiBC what happens to it
   • It oscillates in cyanobacteria, but won't in E. coli b/c transcription isn't controlled
2. What happens when we put a plasmid in cyanobacteria? Does it replicate?
   • Only if it is based on an endogenous plasmid. Paper from 20 yrs back...
   • There are endogenous plasmids (2)
   • PCC7942 has homologous recombination
     • But we don't know if a plasmid will be maintained
     • new: hybrid plasmid possible on this paper
     • Another hybrid plasmid developed by Professor Golden in 1983: link here
3. Do we know what the sigma factor is in Kondo et al
   • We can put in alot
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?
8. How to grow WH8102?
9. Difference in circadian clock b/t PCC7942 and PCC6903?
10. Growth conditions in liquid: use thiiosulfate? Hours light/dark? General streaking?

1. Ask Susan about the plasmids pAM1579 and pAM1303.

Presentations

• Project proposal (week 2)
• File:Cyan week3.ppt
  • Built incubator and obtained WH8102, PCC7942, and PCC6803 strains

Primer Design

IGEM:Harvard/2006/Cyanobacteria/PrimerDesign

• According to BLAST, KaiC (519aa) is only 81% identical between PCC7942 and 6803.
• These papers might have our promoter sequences:

1. Ishiura M, Kutsuna S, Aoki S, Iwasaki H, Andersson CR, Tanabe A, Golden SS, Johnson CH, and Kondo T. Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria. Science. 1998 Sep 4;281(5382):1519-23. DOI:10.1126/science.281.5382.1519 |
2. Kutsuna S, Nakahira Y, Katayama M, Ishiura M, and Kondo T. Transcriptional regulation of the circadian clock operon kaiBC by upstream regions in cyanobacteria. Mol Microbiol. 2005 Sep;57(5):1474-84. DOI:10.1111/j.1365-2958.2005.04781.x |
3. Goto-Seki A, Shirokane M, Masuda S, Tanaka K, and Takahashi H. Specificity crosstalk among group 1 and group 2 sigma factors in the cyanobacterium Synechococcus sp. PCC7942: In vitro specificity and a phylogenetic analysis. Mol Microbiol. 1999 Nov;34(3):473-84. DOI:10.1046/j.1365-2958.1999.01608.x |

• Especially paper #3, which states that there are 4 known sigma factors.
• Paper #2 says that -55 to 14 was experimentally determined to be the promoter/operon area for kaiBC (not kaiA :(); -35 and -10 have homology to sigma 70 in E. coli

Working Team Members

• Hetmann Hsieh (talk)
• Jeffrey Lau (talk)
• Zhipeng Sun (talk)
• David Ramos (talk)