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==Agenda==
{{IGEM:/Harvard/2006/Cyanobacteria}}


For Monday
__TOC__
==Introduction==


*Figure out optimal growing conditions
Welcome to the lab notebook for the Cyanobacteria oscillator project! The goal of our team, composed of four members, is to reconstruct the cyanobacterial circadian oscillator system into E. coli. Three proteins, KaiA, B, and C, have been shown to have an in-vitro phosphorylation state oscillation (Nakajima et al. 2005) by transcriptional-translational independent methods. If this system can be reconstituted in ''E. coli'', there are two important applications:
*Regrow cyanobacteria colonies
*Create Primers (Finish by Tuesday)


==Notebook==
#'''Synthetic Biology''': Creating a functional, oscillating set of proteins is the next logical step from the synthetic "repressilator" system engineered by Elowitz et al. (2000). Although a good proof of concept, the "repressilator" lacks the stability needed from a robust oscillator such as the naturally evolved cyanobacterial oscillator. This robust oscillator could prove useful in an eventual biocircuit.
<calendar>
#'''Circadian Biology''': Cyanobacteria are the simplest model organisms for the study of circadian oscillation. Although circadian oscillation has been fairly well characterized, less is understood at the molecular level. By porting the oscillation system into ''E. coli'', one can begin to understand more precisely the pathways involved in the genomic oscillation of cyanobacteria.
name=/Notebook
date=2006/07/01
view=threemonths
format=%name/%year-%month-%day
weekstart=14
</calendar>


==Reading==
For more background information on the ciracadian system, please check out our "Literature" section. Otherwise, day-to-day work can be found under the "Lab Notebook" tab; we will post major results of our work and links to the days as they become available. If you have questions or comments, feel free to contact us: information is located at the main Harvard iGEM 2006 page. Thanks!
===Cyanobacteria background and practicality===
#[https://dspace.mit.edu/bitstream/1721.1/32302/1/61347289.pdf 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.
# Molecular biology of circadian rhythms / edited by Amita Sehgal
#* Peng's taking a look at it now; in Biolabs
#[http://www-cyanosite.bio.purdue.edu Cyanosite]
#* Lots of information on cyanobacteria culture mediums and good links to other resources.


===Papers on latest findings===
<biblio>
#1 pmid=15831759
#2 pmid=10649285
#3 pmid=16729054
#4 pmid=12727878
</biblio>
# Implementing KaiABC in-vitro and demonstrating circadian oscillation
#* Hetmann's review [[IGEM:Harvard/2006/Brainstorming_Papers_-_Hetmann | here]]
# Good review paper on cyanobacteria oscillator, featuring clearly what is unknown
#* Peng's review [[IGEM:Harvard/2006/Brainstorming_Papers_-_Zhipeng | here]]
# General review paper of current cyanobacteria knowledge recommended by Dave
# Information on measuring Phosphorylated KaiC
#* [[IGEM:Harvard/2006/Xuetal | See here for review from paper + image]]


==Incubator==
Sincerely,<br>
We grow our cyanobacteria in a modified incubator with a timer-controlled fluorescent light. The incubator has a shaking floor.
Zhipeng, Hetmann, Dave, and Jeff
*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 2200-6000 lux.
[[Image:Incubator ext.JPG|thumb|left|Incubator exterior]]
[[Image:Incubator int.JPG|thumb|left|Incubator interior]]
[[Image:Incubator int closeup.JPG|thumb|left|Incubator interior (closeup)]]
<br style="clear:both;"/>


==Cyanobacteria care==
*[http://www.blackwell-synergy.com.ezp1.harvard.edu/doi/abs/10.1111/j.1574-6968.1989.tb03546.x Optimum conditions for growth of cyanobacteria on solid media]
**Contains comparisons of many different media for several strains, including PCC7942 and PCC6803.
*[[Media: Working_with_algae.pdf | Working with algae]]
**General information on growing cyanobacteria
*[[Media: Conversion_lux.pdf | Light conversion guide]]
** Conversion information of units supplied by Peter Weigele.


===Marine (WH8102)===
'''Update 10/27/06:''' We believe we can express the three proteins into e. coli, and that there is interaction between A+C and possible interaction between B+C. See the Lab Notebook for more information.
[http://www-cyanosite.bio.purdue.edu/media/table/SN.html Formula for SN medium]


[http://ccmp.bigelow.org/CI/SN_family.html SN agar]
[[Image:102706_cyanoresult.jpg]]


===Freshwater (PCC7942 and PCC6803)===
==Outline of Findings and Signifigant Dates==
See [[IGEM:Harvard/2006/Protocols/Cyanobacteria|protocols]].
*07/05/06: The incubator for growing up our cyanobacteria is complete; we have cultures growing! [[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-5 | Link]]
*07/10/06: Some computer modeling has been done to see the effect of multiple unsyncronized clocks on phosphorylation state output. [[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-10 | Link]]
*07/21/06: Upon having trouble with site-specific mutagenesis on the KaiA and KaiBC operons from the cyanobacterial genome, we have decided to pursue synthesis of the constructs in parallel with continued extraction attempts. [[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-21 | Link]]
*08/01/06: Preliminary success with site-specific mutagenesis. [[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-1 | Link]]
*08/05/06: Promoter leakness tests come out negative. May have to use low-copy plasmids if we want good control of protein expression in Top10F. [[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-5 | Link]]
*08/11/06: We are moving to the synthetic KaiA, KaiB, and KaiC for future work. [[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-11 | Link]]
*08/30/06: We successfully made the first construct, Lac+RBS+KaiC. [[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-30 | Link]]
*09/01/06: Using the newly developed ligation protocol, we have successfully repeated Lac+RBS+KaiC from 08/30/06 and made Lac+RBS+KaiA. [[IGEM:Harvard/2006/Nicholas_Stroustrup%27s_Notebook#Results_Summary |
Link]]
*10/21/06: Successfully made Lac+RBS+KaiB and Lac+RBS+KaiA+Lac+RBS+KaiC. [[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-10-21 | Link]]
*10/24/06: Successfully made Lac+RBS+KaiB+Lac+RBS+KaiC. [[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-10-24 | Link]]
*10/25/06: Constructs for Stage I have been completed; ready to move to Stage I of Western Blotting, to verify expression of KaiC and interaction of KaiA and KaiB with KaiC. [[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-10-24 | Link]]
*'''10/27/06: Preliminary data indicates that the Kai proteins are being expressed in e. coli and that there is interaction between the three proteins! [[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-10-27 | Link]]'''


== Protocols ==
==Construct Planning==
''Main article: [[IGEM:Harvard/2006/Protocols/Cyanobacteria]]


==Possible Molecular Mechanisms==
[[Image:construct_plans.png|thumb|left|330px|Constructs we plan to create.]]
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===
<br style="clear:both">
[[Image: Barkai_Leibler.jpg |thumb|right| "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)


=== Lengths ===
From VF2 to VR (BioBrick primers):
* KaiA + J04500: 1406 bp
* KaiB + J04500: 859 bp
* KaiC + J04500: 2110 bp


===KaiC phosphorylation model===
[[Image: Xu_2003.jpg |thumb|left| "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.
<br style="clear:both;">


===KaiB spaitotemporal localization model===
[[Image: Kitayamaetal_2003.jpg |thumb|right| "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?


==Agenda==
''See image at right for our long-term project outline.''
[[Image:Cyanobacteria_Flowchart.png|thumb|Long-term project outline]]


===Transcriptional/Translational independent model===
==BioBricks Used==
[[Image: Tomita 2005.jpg |thumb|left| "Transcriptional/translational independent, 2005"]]
*Tomita, Nakajima et al 2005
*Minimal oscillator and an extended timing system
*Best oscillation system currently developed
<br style="clear:both;">
 
===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


==Possible Project Ideas==
:*<bbpart>BBa_J04450</bbpart>
==Important people to contact (/stalk)==
:**RFP device
# Prof. Alexander van Oudenaarden MIT
:**Insert size: 1069bp
#* ''Emailed'', we can meet up with him when he gets back from Woods Hole first week of July
:**[[http://parts.mit.edu/registry/index.php/Part:pSB1A2 pSB1A2]]
#* May have many of the genes we want already on plasmids
:***High-copy, Amp<sup>R</sup>
# Jeffrey Chabot
:***Size: 2079bp
#* Post Doc who wrote the PhD thesis on cyanobacteria
:*<bbpart>BBa_J04500</bbpart>
#* Probably knows a lot of information but I can't find his contact
:**Lac promoter + RBS
# Prof. Susan Golden Texas A&M
:**Insert size: 220bp
#* Is working on pretty much the same stuff we are looking at
:**[[http://parts.mit.edu/registry/index.php/Part:pSB1AK3 pSB1AK3]]
# Peter Weigele MIT
:***High-copy, Amp<sup>R</sup>, Kan<sup>R</sup>
#* Post Doc who works with cyanobacteria
:***Insert size: 3189bp
#* ''Have emailed'' correspondance (see Peng); can get PCC7942 strains
:*[[http://parts.mit.edu/registry/index.php/Part:pSB4A3 pSB4A3]]
#* ''Have contacted'' and obtained PCC7942 and PCC6803 (a glucose-digesting strain)
:**Low-copy, Amp<sup>R</sup>
# Prof. Andrew Knoll Harvard OEB
:**Insert size: 3339 bp
#* Worked with evolutionary cyanobacteria
:*<bbpart>BBa_R0010</bbpart> + <bbpart>BBa_E0241</bbpart>
#* ''Called''; gave references to other experts around the area
:**GFP device
# Prof. Stjepko Golubic BU
:**Insert size: 995 bp
#* Prof. Knoll said he knew a lot about cyanobacteria
# Prof. Colleen Cavanaugh Harvard OEB
 
==Questions that we need to ask==
 
# If we put a reporter downstream of kaiBC what happens to it
# 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''
# Do we know what the sigma factor is in Kondo et al
# Possible ways for reporting if oscillation works
# Any other mechanisms?
# How bad is the codon bias problem / would we need to actually mutate parts of the genome to move to E. coli?
# '''Is there any feasible reporter we can have in E. coli?'''
# How to grow WH8102?
# Difference in circadian clock b/t PCC7942 and PCC6903?
# Growth conditions in liquid: use thiiosulfate? Hours light/dark? General streaking?
# Ask Susan about the plasmids pAM1579 and pAM1303.


==Presentations==
==Presentations==
*[[IGEM:Harvard/2006/Presentation_cyano_week2 | Project proposal (week 2)]]
*[[IGEM:Harvard/2006/Presentation_cyano_week2 | Project proposal (week 2)]]
*[[Image:Cyan week3.ppt|Week 3 progress update]]
*[[Media:Cyan_week3.ppt |Week 3 progress update]]
**Built incubator and obtained WH8102, PCC7942, and PCC6803 strains
**Built incubator and obtained WH8102, PCC7942, and PCC6803 strains
*[[Media:Cyano_week4.ppt |Week 4 progress update]]
*[[Media:Cyanobacteria_Presentation_Week_5.ppt |Week 5 progress update, upd. 10:10 7/17]]
*[[Media:Cyanobacteria_Presentation_Week_6.ppt |Week 6 progress update, upd. 10:02 7/24 HH]]
*[[Media:Cyanobacteria_Presentation_Week_7.ppt |Week 7 progress update]]
*[[Media:Cyanobacteria_presentation_Week_8.ppt |Week 8 progress update]]
*[[Media:Cyanobacteria_presentation_Week_9.ppt |Week 9 progress update]]
*[[Media:Cyanobacteria_presentation_Week_10.ppt |Week 10 progress update, 50% complete]]
*''[[Media:Cyanobacteria_final_presentation.ppt |Final Presentation (incomplete)]]'' --old
*''[[Media:final_presentation_draft2.ppt |Final Presentation (complete)]]'' --old
*[[:Image:Cyano presentation.ppt | Jamboree presentation]] (in progress)
**[[:Image:Cyano_presentation_script.doc|Script]] (in progress)
*[[:Image:Cyano poster.ppt | Cyano poster]] (in progress)
==Team Members==
*[[User:Hetmann|Hetmann Hsieh]] ([[User_talk:Hetmann|talk]], [[Special:Contributions/Hetmann|edits]])
*[[User:JeffreyLau|Jeffrey Lau]] ([[User_talk:JeffreyLau|talk]], [[Special:Contributions/JeffreyLau|edits]])
*[[User:Zhipeng Sun|Zhipeng Sun]] ([[User_talk:Zhipeng_Sun|talk]], [[Special:Contributions/Zhipeng_Sun|edits]])
*[[User:DavidRamos|David Ramos]] ([[User_talk:DavidRamos|talk]], [[Special:Contributions/DavidRamos|edits]])


==Working Team Members==
==Recent Changes==
*[[User:Hetmann|Hetmann Hsieh]] ([[User_talk:Hetmann|talk]])
{{Special:Recentchanges/b=IGEM:Harvard/2006/Cyanobacteria&limit=25}}
*[[User:JeffreyLau|Jeffrey Lau]] ([[User_talk:JeffreyLau|talk]])
*[[User:Zhipeng Sun|Zhipeng Sun]] ([[User_talk:Zhipeng_Sun|talk]])
*[[User:DavidRamos|David Ramos]] ([[User_talk:DavidRamos|talk]])

Latest revision as of 04:28, 3 November 2006

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Introduction

Welcome to the lab notebook for the Cyanobacteria oscillator project! The goal of our team, composed of four members, is to reconstruct the cyanobacterial circadian oscillator system into E. coli. Three proteins, KaiA, B, and C, have been shown to have an in-vitro phosphorylation state oscillation (Nakajima et al. 2005) by transcriptional-translational independent methods. If this system can be reconstituted in E. coli, there are two important applications:

  1. Synthetic Biology: Creating a functional, oscillating set of proteins is the next logical step from the synthetic "repressilator" system engineered by Elowitz et al. (2000). Although a good proof of concept, the "repressilator" lacks the stability needed from a robust oscillator such as the naturally evolved cyanobacterial oscillator. This robust oscillator could prove useful in an eventual biocircuit.
  2. Circadian Biology: Cyanobacteria are the simplest model organisms for the study of circadian oscillation. Although circadian oscillation has been fairly well characterized, less is understood at the molecular level. By porting the oscillation system into E. coli, one can begin to understand more precisely the pathways involved in the genomic oscillation of cyanobacteria.

For more background information on the ciracadian system, please check out our "Literature" section. Otherwise, day-to-day work can be found under the "Lab Notebook" tab; we will post major results of our work and links to the days as they become available. If you have questions or comments, feel free to contact us: information is located at the main Harvard iGEM 2006 page. Thanks!


Sincerely,
Zhipeng, Hetmann, Dave, and Jeff


Update 10/27/06: We believe we can express the three proteins into e. coli, and that there is interaction between A+C and possible interaction between B+C. See the Lab Notebook for more information.

Outline of Findings and Signifigant Dates

  • 07/05/06: The incubator for growing up our cyanobacteria is complete; we have cultures growing! Link
  • 07/10/06: Some computer modeling has been done to see the effect of multiple unsyncronized clocks on phosphorylation state output. Link
  • 07/21/06: Upon having trouble with site-specific mutagenesis on the KaiA and KaiBC operons from the cyanobacterial genome, we have decided to pursue synthesis of the constructs in parallel with continued extraction attempts. Link
  • 08/01/06: Preliminary success with site-specific mutagenesis. Link
  • 08/05/06: Promoter leakness tests come out negative. May have to use low-copy plasmids if we want good control of protein expression in Top10F. Link
  • 08/11/06: We are moving to the synthetic KaiA, KaiB, and KaiC for future work. Link
  • 08/30/06: We successfully made the first construct, Lac+RBS+KaiC. Link
  • 09/01/06: Using the newly developed ligation protocol, we have successfully repeated Lac+RBS+KaiC from 08/30/06 and made Lac+RBS+KaiA. Link
  • 10/21/06: Successfully made Lac+RBS+KaiB and Lac+RBS+KaiA+Lac+RBS+KaiC. Link
  • 10/24/06: Successfully made Lac+RBS+KaiB+Lac+RBS+KaiC. Link
  • 10/25/06: Constructs for Stage I have been completed; ready to move to Stage I of Western Blotting, to verify expression of KaiC and interaction of KaiA and KaiB with KaiC. Link
  • 10/27/06: Preliminary data indicates that the Kai proteins are being expressed in e. coli and that there is interaction between the three proteins! Link

Construct Planning

Constructs we plan to create.


Lengths

From VF2 to VR (BioBrick primers):

  • KaiA + J04500: 1406 bp
  • KaiB + J04500: 859 bp
  • KaiC + J04500: 2110 bp


Agenda

See image at right for our long-term project outline.

Long-term project outline

BioBricks Used

  • <bbpart>BBa_J04450</bbpart>
    • RFP device
    • Insert size: 1069bp
    • [pSB1A2]
      • High-copy, AmpR
      • Size: 2079bp
  • <bbpart>BBa_J04500</bbpart>
    • Lac promoter + RBS
    • Insert size: 220bp
    • [pSB1AK3]
      • High-copy, AmpR, KanR
      • Insert size: 3189bp
  • [pSB4A3]
    • Low-copy, AmpR
    • Insert size: 3339 bp
  • <bbpart>BBa_R0010</bbpart> + <bbpart>BBa_E0241</bbpart>
    • GFP device
    • Insert size: 995 bp

Presentations

Team Members

Recent Changes

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21 April 2024

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