IGEM:Harvard/2006/Cyanobacteria: Difference between revisions

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==="Required Reading"===
{{IGEM:/Harvard/2006/Cyanobacteria}}
====Cyanobacteria backround 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.
#[[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.


====Papers on latest findings====
__TOC__
<biblio>
==Introduction==
#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


===Incubator/Supplies===
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:
*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===
#'''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.
====Activator/Repressor====
#'''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.
*Barkai and Leibler 2000
*Modeled after Eukarayotic systems
*Probably not true
**KaiABC vary as activator/repressor
**Transcription/translation not essential (invitro experiment)


[[Image: Barkai_Leibler.jpg |thumb| "Activator-repressor model from 2000, by Chabot 2005"]]
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!


====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.
[[Image: Xu_2003.jpg |thumb| "KaiC phosphorylation model from 2003, by Chabot 2005"]]


====KaiB spaitotemporal localization model====
Sincerely,<br>
*Kitayama et al 2003
Zhipeng, Hetmann, Dave, and Jeff
*Idea that KaiB rotates location from the membrane to cytosol
**Doesn't the in-vitro experiment disprove this?


[[Image: Kitayamaetal_2003.jpg |thumb| "KaiB spaitotemporal localization, 2003"]]


====Transcriptional/Translational independent model====
'''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.
*Tomita, Nakajima et al 2005
*Minimal oscillator and an extended timing system
*Best oscillation system currently developed


[[Image: Tomita 2005.jpg |thumb| "Transcriptional/translational independent, 2005"]]
[[Image:102706_cyanoresult.jpg]]


===Possible Project Ideas===
==Outline of Findings and Signifigant Dates==
===People to contact===
*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]]
===Questions that we need to ask===
*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]]'''


*Q: If we put a reporter downstream of kaiBC what happens to it
==Construct Planning==
*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===
[[Image:construct_plans.png|thumb|left|330px|Constructs we plan to create.]]


[[IGEM:Harvard/2006/Presentation_cyano_week2 | Moved to new page]]
<br style="clear:both">


3000bp, 11cents/base. --> synthetic
=== Lengths ===
From VF2 to VR (BioBrick primers):
* KaiA + J04500: 1406 bp
* KaiB + J04500: 859 bp
* KaiC + J04500: 2110 bp




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


From the paper:
==BioBricks Used==
  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"]]
:*<bbpart>BBa_J04450</bbpart>
:**RFP device
:**Insert size: 1069bp
:**[[http://parts.mit.edu/registry/index.php/Part:pSB1A2 pSB1A2]]
:***High-copy, Amp<sup>R</sup>
:***Size: 2079bp
:*<bbpart>BBa_J04500</bbpart>
:**Lac promoter + RBS
:**Insert size: 220bp
:**[[http://parts.mit.edu/registry/index.php/Part:pSB1AK3 pSB1AK3]]
:***High-copy, Amp<sup>R</sup>, Kan<sup>R</sup>
:***Insert size: 3189bp
:*[[http://parts.mit.edu/registry/index.php/Part:pSB4A3 pSB4A3]]
:**Low-copy, Amp<sup>R</sup>
:**Insert size: 3339 bp
:*<bbpart>BBa_R0010</bbpart> + <bbpart>BBa_E0241</bbpart>
:**GFP device
:**Insert size: 995 bp
 
==Presentations==
*[[IGEM:Harvard/2006/Presentation_cyano_week2 | Project proposal (week 2)]]
*[[Media:Cyan_week3.ppt |Week 3 progress update]]
**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]])
 
==Recent Changes==
{{Special:Recentchanges/b=IGEM:Harvard/2006/Cyanobacteria&limit=25}}

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

List of abbreviations:
N
This edit created a new page (also see list of new pages)
m
This is a minor edit
b
This edit was performed by a bot
(±123)
The page size changed by this number of bytes

25 April 2024

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