Chiba/Sandbox/Home2

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[[Chiba|Introduction]] | [[Chiba/Project_Design|Project Design]] ( [[Chiba/Engeneering_Flagella|1.Affinity Tag]] | [[Chiba/Communication|2.Communication Module]] | [[Chiba/Quorum_Sensing|3.Size Control]] ) |  [[Chiba/Making Marimo|Making Marimos]] |  [[Chiba/Goal|Our Goal]]<br>
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[[Chiba/Introduction|Introduction]] | [[Chiba/Project_Design|Project Design]] ( [[Chiba/Engeneering_Flagella|1.Affinity Tag]] | [[Chiba/Communication|2.Communication Module]] | [[Chiba/Quorum_Sensing|3.Size Control]] ) |  [[Chiba/Making Marimo|Making Marimos]] |  [[Chiba/Goal|Our Goal]]<br>
[[Chiba/Acknowledgements|Acknowledgements]] | [[Chiba/Team_Members|Team Members]] | [http://chem.tf.chiba-u.jp/igem/ iGEM Chiba Website] | [[Chiba/Members_Only|メンバ連絡簿]]  
[[Chiba/Acknowledgements|Acknowledgements]] | [[Chiba/Team_Members|Team Members]] | [http://chem.tf.chiba-u.jp/igem/ iGEM Chiba Website] | [[Chiba/Members_Only|メンバ連絡簿]]  
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|}
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==Chiba's Project==
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==Project Overview==
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Our iGEM project is to make a '''Marimo-ish gathering of bacteria'''. Marimo is a green spherical shaped algae (shown in Fig.1), which is a popular living organism in Japan as a National Treasure.(<because of its shape?)
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[[Image:Chiba_marimobig.jpg|frame|''Fig1.''Marimo in the lake]]Our iGEM project is to make a '''Marimo-ish gathering of bacteria'''. Marimo is a green spherical shaped algae (shown in Fig.1), which is a popular living organism in Japan as a National Treasure because of its beautiful shape and its smoothness.
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Motivation.<br>
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===Why We Make a Marimo : Spherical Multicellular Organism!? ===
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(more...)
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When you see a shape in Nature, you will notice whether a sphere, which is absolutely symmetric in 3D, is really stable or not in Nature.
 +
In fact, an oil droplet is a sphere in water. Red blood cell in a hypotonic solution shows its shape change to a spherical balloon.  
 +
However multicellular organisms have their shape different from a sphere except Marimo. Of course, other algae do not show spherical shape, they live on a surface of stone.
 +
It is quite intriguing how Marimo remains its spherical shape in a lake!
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==Design==
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Our team focuses understanding how such spherical structure can be sustained even when it Is multicellular organisms.<br>
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Our marimo bacteria requres as follows:
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[[Chiba/Introduction|more...]]
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{| border="1"
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===How To Make Our Marimo===
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| [[Image:Chiba_stickbacteria.png]]
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[[Image:Chiba_marimosystem.png|frame|'''Fig2.''' Our marimo system.]]What we require to our system is as follows:
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|  
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#Affinity Tag.
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'''1.Affinity Tag.'''
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#Communication Module.
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Make a His-tagged Flagella. We aimed to stick bacteria by displaying histidines (which bonds each other through metal ions) on the flagellar filament.
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#Size Control.
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Two cells are used in our system: AHL senders and receivers.  Senders generates the affinity tags constitutively, while receivers generates them only when they are induced by AHL. The marimo-forming goes like this:
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| [[Image:Chiba_prj_com.png]]
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*Make the sender core by sticking with the affinity tag.
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|
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*Insert the sender core into the receiver culture.
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'''2.Communication Module.'''
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*The sender core produces AHL, which make the near receivers to generate the affinity tags and GFPs.
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Make a Bacterial Communication. We make 2 types of cell(sender&receiver) having different gene circuit. Senders sticking each other in advance send a signal to receivers and receivers grow affinity tags.
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*The affinity tagged receiver sticks with the central sender core. This will continue until the AHL cannnot reach the marimo boundary,
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|-
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*When the AHL reached the marimo boundary, the adsorping stops, which makes a finite-sized marimo bacteria.
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| [[Image:Chiba_proj_ahlconc.png]]
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'''3.Size Controler.'''
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Make an AHL localized region for quorum sensing.
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これを組み合わせて以下のように毬藻をつくる：
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==Experiments Overview==
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==Achievements Overview==
 
===[[Chiba/Engeneering_Flagella|Making Affinity Tags]]===
===[[Chiba/Engeneering_Flagella|Making Affinity Tags]]===
[[Image:Chiba_flichisgene.png|frame|'''Fig.''' The short peptide with six histidine (“His-Tag”) was inserted into the fliC D3 domain.]]
[[Image:Chiba_flichisgene.png|frame|'''Fig.''' The short peptide with six histidine (“His-Tag”) was inserted into the fliC D3 domain.]]
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===Making Communication Module===
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===[[Chiba/Communication|Making Communication Module]]===
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#Making Senders
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#Making Receivers
#*[[Image:Chiba_check.png]] AHL > GFP generator (constitutive aiiA) : BBa_T729006
#*[[Image:Chiba_check.png]] AHL > GFP generator (constitutive aiiA) : BBa_T729006
#*[[Image:Chiba_check.png]] AHL > GFP & aiiA generator : BBa_I729005
#*[[Image:Chiba_check.png]] AHL > GFP & aiiA generator : BBa_I729005
#*[[Image:Chiba_check.png]] Sensitive AHL > GFP generator : BBa_I729004
#*[[Image:Chiba_check.png]] Sensitive AHL > GFP generator : BBa_I729004
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[edit]
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#*[[Image:Chiba_nocheck.png]] inverter-aiiA
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Future Work
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#Making Senders
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inverter-aiiA
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#*[[Image:Chiba_nocheck.png]] MetK Sender : Could not deposit to registry
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[[Chiba/Communication|more...]]
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#*1.---
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#*2.---
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#Making Receivers
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#*1.---
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#*2.---
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#*
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#*
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===Controlling Size===
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===[[Chiba/Quorum_Sensing|Controlling Size]]===
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[[Image:MLuxR-test.gif|frame|'''Fig.''' Improved Receiver.]]
#Improve Sender
#Improve Sender
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#*前駆体合成酵素metKを入れてみた．＞結果？
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#*[[Image:Chiba_check.png]] Overexpressed the metK (the AHL precursur synthesis enzyme) in the hope to increase AHL synthesis. : Turned out not to work
#Improve Receiver
#Improve Receiver
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#*LuxRの感度が良くなるmutation（論文より）を入れてみた．＞結果
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#*[[Image:Chiba_check.png]] Inserted 2 mutations in LuxR which is known from the paper to increase activity.
#Localize AHL
#Localize AHL
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#*aiiAその１の回路（〜〜になるはず）＞結果
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#*[[Image:Chiba_check.png]] Tested the GFP expression in constitutive aiiA generator receiver : Went too much.
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#*aiiAその２の回路
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#*[[Image:Chiba_check.png]] Tested the GFP expression in AHL-induced aiiA generator receiver : Needed to twist the circuit more.
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#*その３
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#*[[Image:Chiba_nocheck.png]] AHL-induced inverter aiiA receiver. Not yet assembled.
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===Making Marimo===
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[[Chiba/Quorum_Sensing|more...]]
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==Remaining Task(Work) ==
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===[[Chiba/Making Marimo|Making Marimo]]===
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#[[Image:Chiba_nocheck.png]] Moving FliC-His generator. : Not yet assembled.
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#[[Image:Chiba_nocheck.png]] FliC-his biobrick : Not yet assembled.
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[[Chiba/Making Marimo|more...]]
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==Our Goal==
 
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==[[Chiba/Goal|Our Goal]]==
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Below we describe the goal to make a marimo bacteria.
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*A test of adsorption between flagella
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*A test to confirm a limit of size
 +
*A test to form Marimo
 +
*A test of size control
 +
===Advanced Goal in Our Future ===
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See below link: our brainstorming of the marimo future!
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==Future==
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[[Chiba/Goal|more...]]

Latest revision as of 04:58, 27 October 2007

Chiba logo.png

Introduction | Project Design ( 1.Affinity Tag | 2.Communication Module | 3.Size Control ) | Making Marimos | Our Goal
Acknowledgements | Team Members | [http://chem.tf.chiba-u.jp/igem/ iGEM Chiba Website] | メンバ連絡簿

Project Overview

Fig1.Marimo in the lake
Our iGEM project is to make a Marimo-ish gathering of bacteria. Marimo is a green spherical shaped algae (shown in Fig.1), which is a popular living organism in Japan as a National Treasure because of its beautiful shape and its smoothness.

Why We Make a Marimo : Spherical Multicellular Organism!? 

When you see a shape in Nature, you will notice whether a sphere, which is absolutely symmetric in 3D, is really stable or not in Nature.

In fact, an oil droplet is a sphere in water. Red blood cell in a hypotonic solution shows its shape change to a spherical balloon. However multicellular organisms have their shape different from a sphere except Marimo. Of course, other algae do not show spherical shape, they live on a surface of stone. It is quite intriguing how Marimo remains its spherical shape in a lake!

Our team focuses understanding how such spherical structure can be sustained even when it Is multicellular organisms.
more...

How To Make Our Marimo

Fig2. Our marimo system.
What we require to our system is as follows:
  1. Affinity Tag.
  2. Communication Module.
  3. Size Control.

Two cells are used in our system: AHL senders and receivers. Senders generates the affinity tags constitutively, while receivers generates them only when they are induced by AHL. The marimo-forming goes like this:

  • Make the sender core by sticking with the affinity tag.
  • Insert the sender core into the receiver culture.
  • The sender core produces AHL, which make the near receivers to generate the affinity tags and GFPs.
  • The affinity tagged receiver sticks with the central sender core. This will continue until the AHL cannnot reach the marimo boundary,
  • When the AHL reached the marimo boundary, the adsorping stops, which makes a finite-sized marimo bacteria.

Experiments Overview

Making Affinity Tags

Fig. The short peptide with six histidine (“His-Tag”) was inserted into the fliC D3 domain.
  1. Make the affinity tag by inserting the his-tag into the flagellar fillament.
    • Chiba check.png Sequence confirmed
    • Chiba check.png Swarm confirmed
    • Chiba check.png Flagella strained with anti-flagella antibody
    • Chiba check.png Phenotype confirmed
    • Chiba check.png Affnity confirmed

more...

Making Communication Module

  1. Making Receivers
    • Chiba check.png AHL > GFP generator (constitutive aiiA) : BBa_T729006
    • Chiba check.png AHL > GFP & aiiA generator : BBa_I729005
    • Chiba check.png Sensitive AHL > GFP generator : BBa_I729004
    • Chiba nocheck.png inverter-aiiA
  2. Making Senders
    • Chiba nocheck.png MetK Sender : Could not deposit to registry

more...

Controlling Size

Fig. Improved Receiver.
  1. Improve Sender
    • Chiba check.png Overexpressed the metK (the AHL precursur synthesis enzyme) in the hope to increase AHL synthesis. : Turned out not to work
  2. Improve Receiver
    • Chiba check.png Inserted 2 mutations in LuxR which is known from the paper to increase activity.
  3. Localize AHL
    • Chiba check.png Tested the GFP expression in constitutive aiiA generator receiver : Went too much.
    • Chiba check.png Tested the GFP expression in AHL-induced aiiA generator receiver : Needed to twist the circuit more.
    • Chiba nocheck.png AHL-induced inverter aiiA receiver. Not yet assembled.

more...

Making Marimo

  1. Chiba nocheck.png Moving FliC-His generator. : Not yet assembled.
  2. Chiba nocheck.png FliC-his biobrick : Not yet assembled.

more...


Our Goal

Below we describe the goal to make a marimo bacteria.

  • A test of adsorption between flagella
  • A test to confirm a limit of size
  • A test to form Marimo
  • A test of size control

Advanced Goal in Our Future

See below link: our brainstorming of the marimo future!

more...

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