Chiba/Sandbox/Home2

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<html><link rel="stylesheet" href="/igem07/index.php?title=User:Maiko/Chiba.css&action=raw&ctype=text/css" type="text/css" /></html>
[[Image:chiba_logo.png|center]]
[[Image:chiba_logo.png|center]]
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{| style="border:0;width:100%;" cellpadding="20px" cellspacing="0"
{| style="border:0;width:100%;" cellpadding="20px" cellspacing="0"
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[[Chiba/Introduction|Introduction]] || [[Chiba/Project_Design|Project Design]] | [[Chiba/Engeneering_Flagella|Engeneering Flagella]] | [[Chiba/Quorum_Sensing|Quorum Sensing]] || [[Chiba/Goal|Our Goal]] || [[Chiba/Team_Members|Team Members]]
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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>
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[[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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==Concept==
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==Project Overview==
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[[Image:chiba_marimobig.jpg|frame|Marimo in the lake]]
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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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Chiba University iGEM07 team's project is to make a '''''Marimo-ish gathering of bacteria'''''. ''Marimo'' is a green spherical shaped algae, which is a popular living organism in Japan as a National Treasure. When you come to Japan, you absolutely come across to Marimo in Aquarisms, TV programs or Mangas :)
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===Why We Make a Marimo : Spherical Multicellular Organism!? ===
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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!
 +
Our team focuses understanding how such spherical structure can be sustained even when it Is multicellular organisms.<br>
 +
[[Chiba/Introduction|more...]]
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===How Marimos are Made===
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===How To Make Our Marimo===
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球形の毬藻は，他の崩れた毬藻断片を核として放射状に伸びることによって出来る．よって成長後の球形毬藻は全て一様で核が存在しない．ちなみに最初の無核球形毬藻は，他の泥や砂などを核として成長し崩れた毬藻断片を核としたと考えられている．（イメージ図）
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[[Image:Chiba_marimosystem.png|frame|'''Fig2.''' Our marimo system.]]What we require to our system is as follows:
 +
#Affinity Tag.
 +
#Communication Module.
 +
#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.
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[[Chiba/Marimo|more about marimos...]]
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==Experiments Overview==
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References:
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===[[Chiba/Engeneering_Flagella|Making Affinity Tags]]===
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*Yoshio Sakai, Marimo no Kagaku, 北海道大学図書刊行会, 1991
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[[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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#Make the affinity tag by inserting the his-tag into the flagellar fillament.
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#*[[Image:Chiba_check.png]] Sequence confirmed
 +
#*[[Image:Chiba_check.png]] Swarm confirmed
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#*[[Image:Chiba_check.png]] Flagella strained with anti-flagella antibody
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#*[[Image:Chiba_check.png]] Phenotype confirmed
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#*[[Image:Chiba_check.png]] Affnity confirmed
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[[Chiba/Engeneering_Flagella|more...]]
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<br clear="all">
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===[[Chiba/Communication|Making Communication Module]]===
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#Making Receivers
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#*[[Image:Chiba_check.png]] AHL > GFP generator (constitutive aiiA) : BBa_T729006
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#*[[Image:Chiba_check.png]] AHL > GFP & aiiA generator : BBa_I729005
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#*[[Image:Chiba_check.png]] Sensitive AHL > GFP generator : BBa_I729004
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#*[[Image:Chiba_nocheck.png]] inverter-aiiA
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#Making Senders
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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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===Why Make a "Marimo Bacteria"?===
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===[[Chiba/Quorum_Sensing|Controlling Size]]===
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大腸菌は固体上やプレート上でそれぞれバイオフィルムやコロニーを形成するなど，固体上で集合体を生じることは知られている．しかし液体中で細胞自身が集合する例は調べる限りでは見当たらない．我々は大腸菌のみで毬藻のような独立した有限の形の集合体をつくることを目指した．
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[[Image:MLuxR-test.gif|frame|'''Fig.''' Improved Receiver.]]
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#Improve Sender
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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
 +
#*[[Image:Chiba_check.png]] Inserted 2 mutations in LuxR which is known from the paper to increase activity.
 +
#Localize AHL
 +
#*[[Image:Chiba_check.png]] Tested the GFP expression in constitutive aiiA generator receiver : Went too much.
 +
#*[[Image:Chiba_check.png]] Tested the GFP expression in AHL-induced aiiA generator receiver : Needed to twist the circuit more.
 +
#*[[Image:Chiba_nocheck.png]] AHL-induced inverter aiiA receiver. Not yet assembled.
 +
[[Chiba/Quorum_Sensing|more...]]
 +
===[[Chiba/Making Marimo|Making Marimo]]===
 +
#[[Image:Chiba_nocheck.png]] Moving FliC-His generator. : Not yet assembled.
 +
#[[Image:Chiba_nocheck.png]] FliC-his biobrick : Not yet assembled.
 +
[[Chiba/Making Marimo|more...]]
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==Project==
 
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===What our system requires===
 
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#Adhesives of Bacteria: make a '''His-tagged Flagella'''
 
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[[Image:Chiba_stickbacteira.png]]
 
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#Size Controller: Generate '''AHL Consentration''' in Quorum Sensing
 
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（絵）
 
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===Genetic Circuit===
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==[[Chiba/Goal|Our Goal]]==
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[[Image:Chiba_marimosystem.png]]
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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!
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====Sender====
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[[Chiba/Goal|more...]]
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====Reciever====
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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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