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

25 January 2020

     14:37  Cdominguez Week 2‎‎ 8 changes history+1,130[Cdominguez‎ (8×)]
     
14:37 (cur | prev) +628 Cdominguez talk contribs →‎Conclusion: add
     
14:29 (cur | prev) +100 Cdominguez talk contribs →‎Methods/Results: answered C7
     
14:24 (cur | prev) +119 Cdominguez talk contribs →‎Methods/Results: answer C3
     
14:15 (cur | prev) +53 Cdominguez talk contribs →‎Methods/Results: image 5
     
14:13 (cur | prev) +52 Cdominguez talk contribs →‎Methods/Results: image 4
     
14:12 (cur | prev) +53 Cdominguez talk contribs →‎Methods/Results: image 3
     
14:10 (cur | prev) +62 Cdominguez talk contribs →‎Methods/Results: added image 2
     
14:09 (cur | prev) +63 Cdominguez talk contribs →‎Methods/Results: added image 1
     14:14  (Upload log)[Cdominguez‎ (5×)]
     
14:14 Cdominguez talk contribs uploaded File:CDPicture5.png
     
14:13 Cdominguez talk contribs uploaded File:CDPicture4.png
     
14:11 Cdominguez talk contribs uploaded File:CDPicture3.png
     
14:09 Cdominguez talk contribs uploaded File:CDPicture2.png
     
14:07 Cdominguez talk contribs uploaded File:CDPicture1.png

24 January 2020

     22:52  (User creation log)[Yar‎ (4×)]
     
22:52 User account Arauch talk contribs was created by Yar talk contribs and password was sent by email ‎
     
12:40 User account Advancellsindia1 talk contribs was created by Yar talk contribs and password was sent by email ‎
     
12:40 User account Chasenneedham talk contribs was created by Yar talk contribs and password was sent by email ‎
     
12:40 User account Doreen Cunningham talk contribs was created by Yar talk contribs and password was sent by email ‎
     13:52  Beauchamp:Unwarping‎‎ 3 changes history+1,322[Meghanr‎ (3×)]
     
13:52 (cur | prev) +248 Meghanr talk contribs →‎FSL: TOPUP and FUGUE/FEAT
     
13:50 (cur | prev) +870 Meghanr talk contribs →‎FSL: TOPUP followed by either applytopup or eddy
     
13:35 (cur | prev) +204 Meghanr talk contribs →‎AFNI: unWarp_epi.py
     13:17  BioMicroCenter:BioinfoClasses diffhist +495 Duanduan Ma talk contribs
     12:57  Beauchamp:RAVE‎‎ 3 changes history+82[Michael S Beauchamp‎ (3×)]
     
12:57 (cur | prev) +3 Michael S Beauchamp talk contribs
     
12:56 (cur | prev) +14 Michael S Beauchamp talk contribs
     
12:56 (cur | prev) +65 Michael S Beauchamp talk contribs
     12:08  CHIP:Members‎‎ 6 changes history+72[Gabor Balazsi‎ (6×)]
     
12:08 (cur | prev) +71 Gabor Balazsi talk contribs
     
12:03 (cur | prev) -1 Gabor Balazsi talk contribs
     
12:02 (cur | prev) -152 Gabor Balazsi talk contribs
     
12:01 (cur | prev) +184 Gabor Balazsi talk contribs
     
11:58 (cur | prev) +99 Gabor Balazsi talk contribs
     
11:56 (cur | prev) -129 Gabor Balazsi talk contribs
     09:17  Silver: 2020 Meeting Schedule‎‎ 2 changes history+1[PamSilver‎ (2×)]
     
09:17 (cur | prev) -5 PamSilver talk contribs →‎February
     
09:16 (cur | prev) +6 PamSilver talk contribs →‎January
     08:33  Beauchamp:Orientation diffhist +65 Michael S Beauchamp talk contribs

23 January 2020

     17:43  Jmenzago Week 2‎‎ 7 changes history+585[Jmenzago‎ (7×)]
     
17:43 (cur | prev) +141 Jmenzago talk contribs →‎Acknowledgements: added homework partner names
     
17:38 (cur | prev) +72 Jmenzago talk contribs →‎Results: centered text in table
     
17:25 (cur | prev) +87 Jmenzago talk contribs →‎Identification of alleles
     
17:22 (cur | prev) +81 Jmenzago talk contribs →‎Methods: added two subsections
     
17:21 (cur | prev) +127 Jmenzago talk contribs →‎Results: finished superscripts in tables
     
16:42 (cur | prev) +47 Jmenzago talk contribs →‎Results: added some superscripts to Genotype/Phenotype table
     
16:39 (cur | prev) +30 Jmenzago talk contribs →‎Results: added superscripts for alleles in first table
     16:51  Adinulos Week 2 diffhist +1,571 Adinulos talk contribs notes from the class
     16:50  User talk:Kam D. Dahlquist‎‎ 2 changes history+894[Kam D. Dahlquist‎ (2×)]
     
16:50 (cur | prev) +487 Kam D. Dahlquist talk contribs answered Karina's question
     
16:46 (cur | prev) +407 Kam D. Dahlquist talk contribs answered Nicholas' question
     16:50  Carolyne week 2‎‎ 3 changes history+290[Carolyne‎ (3×)]
     
16:50 (cur | prev) +49 Carolyne talk contribs →‎Methods
     
16:49 (cur | prev) +242 Carolyne talk contribs →‎Methods: added information about the Aipotu program to the methods section and included a comment
     
16:39 (cur | prev) -1 Carolyne talk contribs moved the references and acknowledgements sections be above the template invocation
     16:47  Cdominguez Week 2‎‎ 3 changes history+344[Cdominguez‎ (3×)]
     
16:47 (cur | prev) +26 Cdominguez talk contribs →‎Methods/Results: added evolution tab methods
     
16:43 (cur | prev) +113 Cdominguez talk contribs →‎Methods/Results: added predictions
     
16:40 (cur | prev) +205 Cdominguez talk contribs →‎Purpose: added
     16:46  Jennymchua Week 2 Assignment diffhist +53 Jennymchua talk contribs →‎Methods: changed color of font to red