Team:Tokyo Tech/Projects/rose pattern/index.htm

From 2012.igem.org

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PHA other collage
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==CEll-cell other collage==
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===3OC6HSLdependent3OC12HSLproduction===
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K081016 BBa_K081016]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0062 BBa_R0062]
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===Positivefeedbackassay===
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Text
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==PHA other collage==
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125801 BBa_K125801]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125802 BBa_K125802]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125803 BBa_K125803]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125804 BBa_K125804]
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125501 BBa_K125501]
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125501 BBa_K125501]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125503 BBa_K125503]
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125503 BBa_K125503]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125504 BBa_K125504]
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156018 BBa_K156018]
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156018 BBa_K156018]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156022 BBa_K156022]
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156022 BBa_K156022]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156031 BBa_K156031]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156033 BBa_K156033]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156034 BBa_K156034]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156012 BBa_K156012]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156013 BBa_K156013]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156014 BBa_K156014]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156021 BBa_K156021]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156018 BBa_K156018]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156019 BBa_K156019]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156020 BBa_K156020]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156022 BBa_K156022]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156023 BBa_K156023]
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K342001 BBa_K342001]
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K342001 BBa_K342001]
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K934026 BBa_K934026]
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K934026 BBa_K934026]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K089001 BBa_K089001]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K089002 BBa_K089002]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K089003 BBa_K089003]
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[https://2008.igem.org/Team:Hawaii iGEM08_Hawaii]
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[https://2008.igem.org/Team:Virginia iGEM08_Virginia]
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[https://2008.igem.org/Team:tsinghua iGEM08_tsinghua]
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[https://2008.igem.org/Team:Utah State iGEM08_Utah State]
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[https://2009.igem.org/Team:Duke iGEM09_Duke]
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[https://2010.igem.org/Team:Caltech iGEM10_Caltech]
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[https://2010.igem.org/Team:INSA-Lyon iGEM10_INSA-Lyon]
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aaaa
=Introduction=
=Introduction=
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<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;">  
<div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;">  
__TOC__
__TOC__
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=Cell-Cell communication Project OVERVIEW=
 
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==Gene circuit==
 
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In this project, we define the signal which E.coli gives out as the romantic feeling of Romeo and Juliet.
 
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The story which we reproduce is divided into four scenes.
 
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[[File:gene circuit tokyotech.png|500px|thumb|left|fig1,gene circuit over view]]
 
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[[File:gene circuit positive feedback tokyotech.png|390px|thumb|right|fig2,positive feed back]]
 
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[[File:gene circuit band detect system.png|390px|thumb|right|fig3,band detect system]]
 
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[[File:gene circuit suicide tokyotech.png|390px|thumb|right|fig4,suicide machanism]]
 
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[[File:gene circuit juliet suicide tokyotech.png|390px|thumb|right|fig4,suicide machanism]]
 
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<br><br><br><br>
 
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First, in positive feedback circuit, there are two types of E. coli, Cell R(Romeo) and Cell J(Juliet).  In cell R (fig2), production of LasI is stimulated by LuxR/3OC6HSL complex and In cell J (fig2), production of LuxI is stimulated by LasR/3OC12HSL complex. This indicates that 3OC12HSL produced by cell R and 3OC6HSL produced by cell J indirectly stimulate each other’s production. We express the story of "Romeo met Juliet and fell in love instantly." by using "positive feedback circuit" as love which burns vigorously.
 
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<br><br><br><br><br><br><br><br><br><br><br>
 
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Second, "band detect system(fig3)" is a mechanism which controls production of a signal in Cell J body, when the amount of secretion of the signal by Cell R reaches more than fixed. We express the story of "The priest proposes she fake death and wake up 24 hours later.", using a "band detect system" as Juliette's suspended animation .
 
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<br><br><br><br><br><br><br><br><br><br><br>
 
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Third, LacI controls a suicide gene. If Cell R detects reduction of the signal from Cell J, suicide gene separate in Cell R body.
 
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As a result, suicide gene is revealed and the number of individuals of Cell R becomes fewer. Reduction of individuals means reduction of the signal. We express the story of "Romeo took the poison." by using a suicide mechanism as suicide of Romeo.
 
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<br><br><br><br><br><br><br><br><br><br><br>
 
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Forth, by the same system as Cell R, suicide gene is revealed and the number of individuals of Cell R becomes fewer. Reduction of individuals means reduction of the signal. We express the story of "Juliet stabbed herself with the dirk." by using a suicide mechanism as suicide of Juliette.
 
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Finaly, we argued about feasibility by performing a simulation using modeling about whether all the above stories are materialized.
 
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==Result==
 
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In order to make the story of "Romeo and Juliet", we constructed and characterized several important parts and devices. First we characterized (Parts name), both of which are existing parts. Second, we designed and characterized a new (parts name). Third, we designed and characterized a NEW BioBrick device (Parts name),(explain). Forth, we confirmed that (mechanism). Finally, we simulated our project and confirmed the feasibility of our system.
 
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<br><br><br>
 
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=Scene1 Fall in love Positive feedback mechanism=
=Scene1 Fall in love Positive feedback mechanism=

Latest revision as of 20:44, 22 September 2012

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Template
Tokyotechmakerose.png

Contents


Abastract

[1]





References

1.

Achievement

TEXT

File:Tokyotechrommejulistory.gif


Links

Tokyo tech parts

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K934011 BBa_K934011]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K934012 BBa_K934012]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K934013 BBa_K934013]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K934016 BBa_K934016]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K934022 BBa_K934022]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K934023 BBa_K934023]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K934024 BBa_K934024]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K934025 BBa_K934025]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K934026 BBa_K934026]


CEll-cell other collage

3OC6HSLdependent3OC12HSLproduction

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K081016 BBa_K081016]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0062 BBa_R0062]




Positivefeedbackassay

Text

PHA other collage

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125801 BBa_K125801]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125802 BBa_K125802]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125803 BBa_K125803]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125804 BBa_K125804]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125501 BBa_K125501]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125502 BBa_K125502]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125503 BBa_K125503]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125504 BBa_K125504]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156018 BBa_K156018]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K282000 BBa_K282000]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K338003 BBa_K338003]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K338004 BBa_K338004]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156022 BBa_K156022]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156031 BBa_K156031]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156033 BBa_K156033]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156034 BBa_K156034]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156012 BBa_K156012]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156013 BBa_K156013]


[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156014 BBa_K156014]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156021 BBa_K156021]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156018 BBa_K156018]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156019 BBa_K156019]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156020 BBa_K156020]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156022 BBa_K156022]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156023 BBa_K156023]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K342001 BBa_K342001]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K934026 BBa_K934026]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K089001 BBa_K089001]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K089002 BBa_K089002]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K089003 BBa_K089003]

iGEM08_Hawaii

iGEM08_Virginia

iGEM08_tsinghua

State iGEM08_Utah State

iGEM09_Duke

iGEM10_Caltech

iGEM10_INSA-Lyon aaaa

Introduction

What's PHA

fig1,synthetic mechanism

PHAs are the abbreviation of Polyhydroixyalkanoates, which are linear polyesters produced in nature by bacterial fermentation of sugar and lipids. They are produced by the bacteria to store carbon and energy. PHAs are also a kind of bio-plastics, which are more sustainable because they can be biodegraded in the environment a lot more faster than fossil-fuel plastics. PHBs are the abbreviation of polyhydrobutyrates ,which are a kind of polyhydroalkanoates. In our project we used the poly-3-hydroxybutyrate（P3HB）form of PHBs, which is the most common type of polyhydroxyalkanoate synthesized by the gene phaC1-A-B from Ralstonia eutropha H16.

As long as we know ,there was no team succeeded in synthesizing PHAs in iGEM format before. But this year we are very excited to tell you that we made it and we are going to show it to all of you. 9/4

Project Overview

Pre-experiment

Before the igem format vector coming，we actually synthesized PHA by using the vector PSB1C3 on E.coli JM109.We synthesized PHA by following steps. 1.Take the right gene phaC1-A-B from R.eutropha, and increase it by PCR 2.Insert the gene into the PSB1C3 vector by using a series of genetic operation including ligation and ethanol precipitation 3. Put the plasmid into E.coli JM109 by using the technique of transformation and plant it on LBmedium with Cm and Nile-Red then culture it overnight. 4. Check whether colonies are stained orange under UV 5. Produce PHAs by quantity by main culture the E.coli 6. Collect the strains and freeze dry it. 7. Stain the product and Measure the weight

Assay In order to check whether the product is PHA or not ,we used several ways of assay. 1. Read the gene sequence of the gene 2. See whether the product can be stained by Nile-Red or Nile-Blue 3. Make sure the product under microscope 9/4

Production of PHB main culture

Protocol

1 Wash Erlenmeyer flasks with mild detergent then rinse with distilled water.

2 Measure 4% LB medium and add it to each Erlenmeyer flask inside clean bench.

3 Add 96% distilled water to each Erlenmeyer flask and cover the flasks with four-folded aluminum foil.

4 Set all flasks into autoclave

5 Clear the clean bench

6 Add 1/1000 Chloramphenicol and 4% Glucose solution (50%) after the medium is completely cooled.

7 Add 2% preculture solution into each flasks and shaking culture at 37 ° C for 72 hours.

Collection of PHBs in JM109

1 Weigh empty 50ml falcon tube without lid and make a record.

2 Add some culture solution into each tube.

3 Set the tubes into centrifuge and make sure that the label faces outside.

4 4 ° C ,5000G ,10min in centrifuge.

5 Remove the supernatant with electric pipettor then add culture solution and set in centrifuge again.

6 After adding all the culture solution and setting in centrifuge, remove the supernatant and add water, set in centrifuge again.

7 Remove the supernatant and add a little amount of water.

8 Cover the tubes with double layers of parafilms and fully freeze them.

Freeze drying

1 Poke several holes on the tubes’ parafilm with toothpick.

2 Set the tubes on the freeze drying machine.

3 Freeze dry for 3 days.

date9/4


Text Text Text Text Text Text Text
Colorblindnesscontentcontentkorekaigyou kaigyou korecontent.#######content.#######
Familial Hypercholesterolemiacontentcontentcontent contentcontent.#######content.#######
Myc-gene CancerTextTextTextText TextText.#######
TextTextTextTextText TextText.#######


Text Text Text Text Text Text Text
Colorblindnesscontentcontentkorekaigyou kaigyou korecontent.#######content.#######
Familial Hypercholesterolemiacontentcontentcontent contentcontent.#######content.#######
Myc-gene CancerTextTextTextText TextText.#######


Gene Circuit


TextA TextA
TextBTextB
TextBTextB
TextBTextB
TextBTextB
TextBTextB
TextBTextBv
TextB-







bar

Tokyotechlogo2012.png

cell-cell communication

Scene1 Fall in love Positive feedback mechanism

LasR generator

fig5,LasR generator
fig5,LasR generator
fig5,LasR generator deta














Text... see more














3OC6HSL→pLuxLasI-PLas

fig5,3OC6HSL→pLuxLasI-PLas
fig5,3OC6HSL→pLuxLasI-PLas
fig5,3OC6HSL→pLuxLasI-PLas deta
















Text... see more






3OC12HSL→pLasLuXI-PLux

fig5,3OC6HSL→pLuxLasI-PLas
fig5,3OC6HSL→pLuxLasI-PLas
fig5,3OC6HSL→pLuxLasI-PLas deta
















Text... see more





positive feedback assay

fig5,3OC6HSL→pLuxLasI-PLas
fig5,3OC6HSL→pLuxLasI-PLas
fig5,3OC6HSL→pLuxLasI-PLas


























Band detect system

Lux-Tet hybrid promoter assay

fig5,Lux-Tet hybrid promoter assay
fig5,Lux-Tet hybrid promoter assay
fig5,Lux-Tet hybrid promoter assay deta













Text... see more








Scene3 Romeo Suicide

Las→Lac assay

fig5,3OC6HSL→pLuxLasI-PLas
fig5,3OC6HSL→pLuxLasI-PLas












Scene4 Juliet Suicide

Lux→Lac assay

fig5,3OC6HSL→pLuxLasI-PLas
fig5,3OC6HSL→pLuxLasI-PLas













Text... see more

modeling

Romeo&Juliette signal change with times

Text







>

Romeo&Juliette Formula

Conclusion

We succeeded in constructing (number) new BioBrick, (name). We characterized mechanism. From the experimental data and simulation result, we confirmed the feasibility of the project "Romeo and Juliette".
Retrieved from "http://2012.igem.org/Team:Tokyo_Tech/Projects/rose_pattern/index.htm"