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

From 2012.igem.org

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Contents


Abastract

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References

1.

Achievement

TEXT

Tokyotechrommejulistory.gif


Links

Tokyo tech parts

BBa_K934001

BBa_K934011

BBa_K934012

BBa_K934013

BBa_K934016

BBa_K934022

BBa_K934023

BBa_K934024

BBa_K934025

BBa_K934026


CEll-cell other collage

3OC6HSLdependent3OC12HSLproduction

BBa_K081016

BBa_R0062




Positivefeedbackassay

Text

PHA other collage

BBa_K125801

BBa_K125802

BBa_K125803

BBa_K125804

BBa_K125501

BBa_K125502

BBa_K125503

BBa_K125504

BBa_K156018

BBa_K282000

BBa_K338003

BBa_K338004

BBa_K156022

BBa_K156031

BBa_K156033

BBa_K156034

BBa_K156012

BBa_K156013


BBa_K156014

BBa_K156021

BBa_K156018

BBa_K156019

BBa_K156020

BBa_K156022

BBa_K156023

BBa_K342001

BBa_K934026

BBa_K089001

BBa_K089002

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


Gene Circuit


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Tokyotechlogo2012.png

cell-cell communication

Scene1 Fall in love Positive feedback mechanism

LasR generator

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














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3OC6HSL→pLuxLasI-PLas

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
















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3OC12HSL→pLasLuXI-PLux

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
















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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













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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













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modeling

Romeo&Juliette signal change with times

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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".