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===Peng's Background List===
{{IGEM:/Harvard/2006/Cyanobacteria}}
[https://dspace.mit.edu/bitstream/1721.1/32302/1/61347289.pdf Genetic noise in the cyanobacterial oscillator]


===Incubator/Supplies===
__TOC__
*Incubator Dimensions: 20 x 20 in.
==Introduction==
*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.


===Possible Molecular Mechanisms===
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:
====Activator/Repressor====
*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"]]
#'''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.
#'''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.


====KaiC phosphorylation model====
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!
*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====
*Kitayama et al 2003
*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"]]
Sincerely,<br>
Zhipeng, Hetmann, Dave, and Jeff


====Transcriptional/Translational independent model====
*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"]]
'''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.


===Possible Project Ideas===
[[Image:102706_cyanoresult.jpg]]
===People to contact===
===Questions that we need to ask===


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


===Project Presentation===
==Construct Planning==


3000bp, 11cents/base. --> synthetic
[[Image:construct_plans.png|thumb|left|330px|Constructs we plan to create.]]


[[Image: Presentation.ppt | "Project Proposal Presentation (The triple P)"]]
<br style="clear:both">


Think about these questions when preparing your project proposals for the group meeting.
=== Lengths ===
From VF2 to VR (BioBrick primers):
* KaiA + J04500: 1406 bp
* KaiB + J04500: 859 bp
* KaiC + J04500: 2110 bp


For each project idea:


* '''What is the specific goal of the project?'''
**Populate Biobricks catalog
**Biobrick a KaiABC oscillator (for use in cyanobacteria AND/OR e. coli)
***[http://www.kazusa.or.jp/cyano/WH8102/cgi-bin/orfinfo.cgi?title=Chr&name=SYNW0548&iden=1 This site] shows the location of the kaiABC genes in WH8102 strain. 2.866kb for kaiABC + non-coding region.
***Research shows that the KaiABC proteins alone will oscillate ''in vitro'' (Nakajima et al. 2005)
**Test the oscillator in E. coli to create a "nightlight"
***Use a luciferase gene reporter, which was done in (Kondo et al. 2000)
***Also can measure KaiC activity; create a chimeric protein w/GFP
**Synthesis of ~3kb KaiABC w/ codon replacement of Ala of Leu to use in E. coli
***Estimated cost is $0.11/bp w/o error correction; $2/bp with error correction (Tian et. al 2004)
****But the Church lab has a better way of doing this?
***DNA synthesis provides a backup in case direct insert of KaiABC into E. coli fails
***There is a known codon bias problem with 2 amino acids (can't find source but I found it the other day): then, we can synthetically modify the codons for these 2 aa's to be compatible in e. coli
** '''Alternate phrasing, courtesy of Kit Parker - what is the "deliverable?" The thing you will point to and say "this is our project?"'''
***Our deliverable is a BioBrick part(s)
* '''What are two or three possible means of implementing the idea?'''
**Biobrick the cyanobacteria KaiABC
**Insert directly into E. coli to create a "nightlight"
**Synthesize E. coli compatible KaiABC and implement in E. coli
**Create a circuit with other BioBricks
**Last resort: Just create a cyanobacteria "nightlight" if all E. coli steps fail
* '''Risk'''
** '''How many untested things have to work for the project to succeed?'''
***Should work unless something in E. coli causes it not to
****Reporter gene should have no problem
***Codon bias may be a problem
***More proteins may be involved than KaiABC
****But KaiABC have been shown to work ''in vitro''
***Transcription regulation of the KaiABC proteins
****We know that KaiA mRNA remains constant as KaiC fluctuates (Wang et. al 2005)
** '''How will you test whether those things work or not?'''
***If we don't get results / alternative methods such as synthesis
** '''How will you adjust your plan when one of these things fails to work?'''
***We have backup plans, such as only implementing a "nightlight" in cyanobacteria
** '''How will you minimize the time/effort/resources lost to a failed design?'''
*** '''Can your time/effort/resources apply to more than one design simultaneously?'''
* '''Reward'''
** '''How cool, fun, exciting is the project for you?'''
***It's cool, fun, AND exciting!
** '''What if any is the usefulness or societal benefit of the project?'''
***Clock oscillator
****Can experimentally vary the period of the oscillator from 14h to 60h (Kondo et. al 2000) with KaiC point mutations
****Can further discretise by half
***A bacterial "timer"
***Could be used as a clock for gene circuits, analogous to a clock signal ''in silico'' (but may be too slow)
***Nightlight
** '''What is going to impress the judges in November?'''
*** Biobricks part!
* '''Timeline'''
** '''What are the project milestones? (design, construction, testing)'''
**#Getting ''WH8102'' strain of cyanobacteria ''1-2 wks''
**#*Prof. Wang at Yale wrote a review, so he may know how to obtain this strain - we will contact him
**#*Otherwise we may have to take a field trip to tour Japan, or check papers for sources
**#*'''EDIT''': Strain PCC7942 works also; MIT says it is the model system for studying circadian rhythm; [http://cyano.genome.jp/cgi-bin/CYORF_open_Search_list.pl?OPE_ITEM=near_gene&OPE_VALUE=5&ANNID=Synpcc7942_1218&SEARCH_ITEM=accession&ORG_TYPE=syf&ORG=syf] has the location for KaiA, B, C. Will email people for these two strains.
**#Creating a cyanobacteria biobrick / extracting KaiABC genes ''1-2 wks''
**#*Designing primers can be done beforehand
**#Designing a feasible E. coli version of KaiABC (can be done simultaneously with step 1) ''1-2 wks''
**#*Research into the necessary modifications
**#*Making the modifications of the 3kb sequence (should be fast)
**#*Send to synthesize
**#Implementing into E. coli both versions ''Long time (5wk+)''
**#*Design either chimeric protein or luciferase (Perry?)
**#*Implementation and testing
** '''What is the estimated time required for each? (always overestimate)'''
** '''If you can't reach your ultimate goal by August, is there a satisfying intermediate goal?'''
***We WILL create a biobricked part that works for cyanobacteria at least
***And if worse comes to worse we'll make a cyanobacteria nightlight
** '''What is the immediate next step in pursuing the project?'''
***See steps 1 and 3 above
*** '''If DNA synthesis will be required, how soon will you have the sequence designed?'''
****1-2 weeks
-----------------------------------


*Nakajima et. al: ''in vitro'', the KaiABC proteins oscillate robustly by themselves, albeit with a lower amplitude than ''in vivo''
==Agenda==
''See image at right for our long-term project outline.''
[[Image:Cyanobacteria_Flowchart.png|thumb|Long-term project outline]]


'''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.
==BioBricks Used==


From the paper:
:*<bbpart>BBa_J04450</bbpart>
  KaiC Phosphorylation Assay. Cyanobacterial cultures were grown to
:**RFP device
  an OD750 of 0.2. After a 12-h dark pulse, the cultures were treated
:**Insert size: 1069bp
  with 10�MIPTGfor 3 h at 30°C in light with air bubbling, and�40
:**[[http://parts.mit.edu/registry/index.php/Part:pSB1A2 pSB1A2]]
  ml of the cultures were collected for preparation of total extracts.
:***High-copy, Amp<sup>R</sup>
  Immunoblot analysis for KaiC was performed on 5 �g of total
:***Size: 2079bp
  proteins per lane according to the previous description (20), except
:*<bbpart>BBa_J04500</bbpart>
  that we used a highly specific mouse polyclonal antibody to KaiC
:**Lac promoter + RBS
  and a SuperSignalWest Pico Chemiluminescent Substrate (Pierce).
:**Insert size: 220bp
  The phosphorylated KaiC signals were quantified as before (16)
:**[[http://parts.mit.edu/registry/index.php/Part:pSB1AK3 pSB1AK3]]
  and analyzed by Student’s t test.
:***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


[[Image: Xu_gelp.jpg |thumb| "Image of figure shown in Xu paper"]]
==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:
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The page size changed by this number of bytes

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