IGEM:Harvard/2006/Cyanobacteria

From OpenWetWare
Jump to navigationJump to search

<html><style type='text/css'> .tabs {

 font-size:80%;
 font-weight:none;
 width: 100%;
 color: #FFFFFF;
 background:#FFFFFF url("/images/5/54/DarkgreenTab-bg.gif") repeat-x bottom;

}

.tabs li {

 background:url("/images/3/36/DarkgeenTab-left.gif") no-repeat left top;

}

.tabs a,.tabs strong {

 background:url("/images/d/d3/DarkgreenTab-right.gif") no-repeat right top;
 color:#FFFFFF;
 padding: 3px 10px 3px 4px;

}

.tabs strong{

 color:#CCFF00;
 background-image:url("/images/b/b1/DarkgreenTab-right_on.gif");

}

.tabs a:hover{

 color:#66FF00;

}


</style></html>


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.

102706 cyanoresult.jpg

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

21 September 2020

     07:26  Yeo lab:news archive diffhist +128 Yoonyeo talk contribs →‎2019
     07:26  Yeo lab diffhist -37 Yoonyeo talk contribs →‎News
N    00:04  User:Bonhivers/Notebook/BIL421/Entry Base diffhist +950 Bonhivers talk contribs Autocreated Lab Notebook name=User:Bonhivers/Notebook/BIL421/Entry Base, content from MediaWiki:EntryContentDefault
N    00:04  User:Bonhivers/Notebook/BIL421 diffhist +1,574 Bonhivers talk contribs Autocreated Lab Notebook name=User:Bonhivers/Notebook/BIL421, content from MediaWiki:ProjectContentDefault
N    00:04  User:Bonhivers/Notebook diffhist +75 Bonhivers talk contribs Autocreated Lab Notebook name=User:Bonhivers/Notebook, content from MediaWiki:NotebookContentDefault

20 September 2020

     22:22  Nathan R Beshai Week 3‎‎ 3 changes history +2,532 [Nathan R. Beshai‎ (3×)]
     
22:22 (cur | prev) +1,750 Nathan R. Beshai talk contribs added 2 figures
     
13:28 (cur | prev) 0 Nathan R. Beshai talk contribs changed spelling
     
13:28 (cur | prev) +782 Nathan R. Beshai talk contribs Finished Introduction
     22:10  Lee:JC‎‎ 2 changes history +58 [Wooin Lee‎ (2×)]
     
22:10 (cur | prev) +35 Wooin Lee talk contribs →‎2020
     
22:04 (cur | prev) +23 Wooin Lee talk contribs →‎2020
     21:52  (Upload log) [Mahesh Chandra Patra‎; Nathan R. Beshai‎ (4×)]
     
21:52 Nathan R. Beshai talk contribs uploaded File:Nathan figure4.png(Added figure 4)
     
21:51 Nathan R. Beshai talk contribs uploaded File:Nathan figure3.png(added figure3)
     
21:50 Nathan R. Beshai talk contribs uploaded File:Nathan figure2.png
     
21:50 Nathan R. Beshai talk contribs uploaded File:Nathan figure1.png(added figure 1)
     
20:10 Mahesh Chandra Patra talk contribs uploaded File:Snk-fig-slash.png
     20:18  Sangdunchoi:company‎‎ 27 changes history +99 [Mahesh Chandra Patra‎ (27×)]
     
20:18 (cur | prev) +33 Mahesh Chandra Patra talk contribs →‎Contact
     
20:18 (cur | prev) +33 Mahesh Chandra Patra talk contribs →‎Notice
     
20:17 (cur | prev) 0 Mahesh Chandra Patra talk contribs →‎20px S&K News
     
20:17 (cur | prev) 0 Mahesh Chandra Patra talk contribs →‎20px R&D
     
20:17 (cur | prev) 0 Mahesh Chandra Patra talk contribs →‎20px Vision
     
20:16 (cur | prev) 0 Mahesh Chandra Patra talk contribs →‎10px About snktherapeutics.com
     
20:16 (cur | prev) 0 Mahesh Chandra Patra talk contribs →‎20px About snktherapeutics.com
     
20:16 (cur | prev) 0 Mahesh Chandra Patra talk contribs →‎20px S&K Therapeutics
     
20:15 (cur | prev) +33 Mahesh Chandra Patra talk contribs →‎S&K News
     
20:15 (cur | prev) -2 Mahesh Chandra Patra talk contribs →‎Target
     
20:14 (cur | prev) +33 Mahesh Chandra Patra talk contribs →‎R&D
     
20:13 (cur | prev) -1 Mahesh Chandra Patra talk contribs →‎20px S&K Therapeutics
     
20:13 (cur | prev) +33 Mahesh Chandra Patra talk contribs →‎Vision
     
20:13 (cur | prev) +1 Mahesh Chandra Patra talk contribs →‎20px S&K Therapeutics
     
20:13 (cur | prev) 0 Mahesh Chandra Patra talk contribs →‎30px About snktherapeutics.com
     
20:12 (cur | prev) 0 Mahesh Chandra Patra talk contribs →‎10px S&K Therapeutics
     
20:12 (cur | prev) +1 Mahesh Chandra Patra talk contribs →‎30pxS&K Therapeutics
     
20:12 (cur | prev) +1 Mahesh Chandra Patra talk contribs →‎30pxAbout snktherapeutics.com
     
20:12 (cur | prev) +32 Mahesh Chandra Patra talk contribs →‎About snktherapeutics.com
     
20:11 (cur | prev) +32 Mahesh Chandra Patra talk contribs →‎S&K Therapeutics
     
01:52 (cur | prev) -25 Mahesh Chandra Patra talk contribs
     
01:50 (cur | prev) +25 Mahesh Chandra Patra talk contribs
     
01:49 (cur | prev) 0 Mahesh Chandra Patra talk contribs →‎S&K Therapeutics
     
01:48 (cur | prev) +4 Mahesh Chandra Patra talk contribs →‎S&K Therapeutics
     
01:48 (cur | prev) -4 Mahesh Chandra Patra talk contribs →‎S&K Therapeutics
     
01:47 (cur | prev) -98 Mahesh Chandra Patra talk contribs
     
01:43 (cur | prev) -32 Mahesh Chandra Patra talk contribs
     08:36  User:Owen R. Dailey‎‎ 4 changes history +92 [Owen R. Dailey‎ (4×)]
     
08:36 (cur | prev) -6 Owen R. Dailey talk contribs →‎Acknowledgements: Fixed a typo
     
08:36 (cur | prev) +28 Owen R. Dailey talk contribs →‎Acknowledgements: Linked my homework partner's user page
     
08:19 (cur | prev) +6 Owen R. Dailey talk contribs →‎Personal Interests and Hobbies: Made the sports teams headers smaller
     
08:18 (cur | prev) +64 Owen R. Dailey talk contribs →‎Sports: Added headers for sports teams
     08:33  Template:Owen R. Dailey‎‎ 3 changes history +765 [Owen R. Dailey‎ (3×)]
     
08:33 (cur | prev) +1 Owen R. Dailey talk contribs →‎Idividual Journals: Corrected a typo in the word "individual"
     
08:33 (cur | prev) +489 Owen R. Dailey talk contribs →‎Class Journals: Added labels
     
08:21 (cur | prev) +275 Owen R. Dailey talk contribs →‎Assignments: Added labels to links

19 September 2020