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

7 June 2023

6 June 2023

     21:20 Upload log ChanEngr talk contribs uploaded File:2023YU Chengdu.JPG
     21:20  X-Lab‎‎ 4 changes history +170 [ChanEngr‎ (4×)]
     
21:20 (cur | prev) +19 ChanEngr talk contribs
     
21:17 (cur | prev) +1 ChanEngr talk contribs
     
21:14 (cur | prev) +35 ChanEngr talk contribs
     
20:46 (cur | prev) +115 ChanEngr talk contribs
     14:52  Eccles: Lab meeting schedule diffhist +37 MikeEccles talk contribs (→‎Lab meeting schedule)
     14:38  CHIP:News diffhist +251 Gabor Balazsi talk contribs
     14:36  CHIP:Talks‎‎ 3 changes history +809 [Gabor Balazsi‎ (3×)]
     
14:36 (cur | prev) +1 Gabor Balazsi talk contribs
     
14:36 (cur | prev) +803 Gabor Balazsi talk contribs
     
14:30 (cur | prev) +5 Gabor Balazsi talk contribs
 m   08:29  User:Richard J. Abbott diffhist 0 Richard J. Abbott talk contribs (→‎Research)
     08:24  User:Olivier Borkowski diffhist +50 Olivier Borkowski talk contribs (→‎Useful links)
 m   08:13  Senecio Research Network‎‎ 3 changes history +806 [Richard J. Abbott‎ (3×)]
 m   
08:13 (cur | prev) +519 Richard J. Abbott talk contribs (→‎Recent Publications)
 m   
07:51 (cur | prev) +8 Richard J. Abbott talk contribs (→‎Recent Publications)
 m   
07:48 (cur | prev) +279 Richard J. Abbott talk contribs (→‎Recent Publications)
     02:15  Y3 BME Automation Group Project : References‎‎ 2 changes history +1,452 [Jyf20‎ (2×)]
     
02:15 (cur | prev) +5 Jyf20 talk contribs (→‎Brochure)
     
02:14 (cur | prev) +1,447 Jyf20 talk contribs (→‎Brochure)

5 June 2023

     20:39 User creation log User account Ruaidhri talk contribs was created by Yar talk contribs and password was sent by email ‎
     19:44 Upload log Xinyu Liu talk contribs uploaded File:AS2023.jpg
     19:41  Gao lab:Publications diffhist +400 Xinyu Liu talk contribs (→‎Publications)

4 June 2023

3 June 2023

     23:44  Lee:JC‎‎ 6 changes history +26 [Wooin Lee‎ (6×)]
     
23:44 (cur | prev) +9 Wooin Lee talk contribs (→‎Schedule)
     
23:23 (cur | prev) −10 Wooin Lee talk contribs (→‎Schedule)
     
23:22 (cur | prev) +6 Wooin Lee talk contribs (→‎Schedule)
     
23:21 (cur | prev) +7 Wooin Lee talk contribs (→‎Schedule)
     
23:01 (cur | prev) +7 Wooin Lee talk contribs (→‎Schedule)
     
22:53 (cur | prev) +7 Wooin Lee talk contribs (→‎Schedule)

1 June 2023

     23:23  Lee:Lab Members‎‎ 4 changes history +169 [Wooin Lee‎ (4×)]
     
23:23 (cur | prev) +164 Wooin Lee talk contribs (→‎Research rotation/internship students (past))
     
23:21 (cur | prev) +26 Wooin Lee talk contribs (→‎Graduate students)
     
23:20 (cur | prev) +31 Wooin Lee talk contribs (→‎Alumni)
     
23:19 (cur | prev) −52 Wooin Lee talk contribs (→‎Graduate students)
     17:46  BioMicroCenter:Pricing diffhist −79 Stuart S. Levine talk contribs (→‎SHORT READ LIBRARY - RNA)
     09:06  UA Biophysics:Protocols:Carotenoid Extration ESP‎‎ 5 changes history +2,390 [Elizabeth Suesca‎ (5×)]
     
09:06 (cur | prev) +53 Elizabeth Suesca talk contribs
     
09:05 (cur | prev) +59 Elizabeth Suesca talk contribs
     
09:04 (cur | prev) +474 Elizabeth Suesca talk contribs
     
08:43 (cur | prev) +781 Elizabeth Suesca talk contribs (→‎Procedimiento)
     
08:25 (cur | prev) +1,023 Elizabeth Suesca talk contribs (→‎Materiales para 6 gramos de célula)
     09:04  (Upload log) [Elizabeth Suesca‎ (2×)]
     
09:04 Elizabeth Suesca talk contribs uploaded File:Cloroformo.png
     
08:25 Elizabeth Suesca talk contribs uploaded File:Acetato.png
     07:49  WAKNA:Journal Club‎‎ 8 changes history +2,612 [Berthold Drexler‎ (8×)]
     
07:49 (cur | prev) +1 Berthold Drexler talk contribs (→‎WAKNA Journal Club)
     
07:48 (cur | prev) +2 Berthold Drexler talk contribs (→‎Die TOP 5 Publikationen aus dem Bereich Neuroanästhesie im Jahr 2022)
     
07:47 (cur | prev) +1 Berthold Drexler talk contribs (→‎WAKNA Journal Club)
     
07:46 (cur | prev) +1 Berthold Drexler talk contribs (→‎Die TOP 5 Publikationen aus dem Bereich Neuroanästhesie im Jahr 2022)
     
07:45 (cur | prev) +4 Berthold Drexler talk contribs (→‎Die TOP 5 Publikationen aus dem Bereich Neuroanästhesie im Jahr 2022)
     
07:45 (cur | prev) +1 Berthold Drexler talk contribs
     
07:44 (cur | prev) +2 Berthold Drexler talk contribs (→‎Die TOP 5 Publikationen aus dem Bereich Neuroanästhesie im Jahr 2022)
     
07:44 (cur | prev) +2,600 Berthold Drexler talk contribs