Chiba

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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|Home]]<br>
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<span style="font-size:120%;font-weight:bold;">[[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]]</span><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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==Introduction==
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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|'''photo. 1''' 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. We borrowed the name ''Marimo'' because of its name publicity (maybe only in Japan, which we didn't notice until recently) and their marvelous velvet which charmed us so much. Our motivations for making ''Marimo bacteria'' is as follows.
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===[[Chiba/Introduction|Introduction: Why We Make a Marimo]]===
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====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.
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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.
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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.
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It is quite intriguing how Marimo remains its spherical shape in a lake!
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===Why Make a "Marimo"?===
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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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'''Making 3D colonies'''- for next generation (lazier) molecular biologists<br>
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[[Chiba/Introduction|more...]]
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For years microbiologists have been using agar plates to isolate cells from each other. By spreading the diluted cells on a solid surface, we can make "colonies", dome-shaped gathering of genetically identical cells. Although convenient, this is only two-dimentional. What if we can create three dimentional (spherical) colonies with controlled/ defined size? Thus we can eliminate the plating process that everybody hates. Combined with the microfluidics devices, we might be able to pick, isolate, count, or innoculate each of the floating yet independent colonies to conduct routine works in future molecular biology. Crazy thought? Well, that is exactly what our advisors say.<br><br>
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'''Toward the control the population size of the bacteria community'''<br>
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===[[Chiba/Project Design|Project Design: How To Make Our Marimo]]===
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Even in the bacteria community, sometimes they need to do the population control. This is especially so when we think about the chemical production using bacteria robots. <br><br>
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[[Image:Chiba_marimosystem.png|frame|'''scheme. 1''' Our marimo system.]]What we require to our system is as follows:
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#Affinity Tag.
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#Communication Module.
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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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*Make the sender core by sticking with the affinity tag.
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*Insert the sender core into the receiver culture.
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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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*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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*When the AHL reached the marimo boundary, the adsorping stops, which makes a finite-sized marimo bacteria.
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[[Chiba/Project Design|more...]]
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Besides the above illogical/ unjustified reasons, we think this project leads to the behavior control of the bacterial comunity. Lots of challenge in the project including diffusion control of small/ large molecules, chemical production/ degradation balancing,.....<br>
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==Experiments Overview==
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==Backgrounds==
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===[[Chiba/Engeneering_Flagella|Making Affinity Tags]]===
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===Marimo?===
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[[Image:Chiba_flichisgene.png|frame|'''scheme. 2''' The short peptide with six histidine (“His-Tag”) was inserted into the fliC D3 domain.]]
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[[Image:Chiba_3marimo.png|frame|'''Fig2.'''Three marimo forms.]] Marimo is known as a spherical shaped algae which could be found, for example, in the Lake Akan in Hokkaido, Japan. The Lake Akan's Marimo is defined as a Natural Tresure of the country, because of its beautiful velvet and its sphrerical shape.  
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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
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#*[[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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Actually, the name "marimo" indicates the algae filament, not the sphere. The sphere shape is one of the three growth forms of the aggregated marimo fillaments. Another growth form lives as ''free-floating fillaments'' as small tufts of unattached filaments that frequently form a carpet on the muddy lake bottom. The third is ''epilithic'' (growing on rocks). Marimos are found in some of the lakes in Japan and other countries, but the ''beautiful spherical shaped'' marimos are only known in Iceland, Estonia and Japan.
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===[[Chiba/Communication|Making Communication Module]]===
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[[Image:BBa I729005 circuit.jpg|frame|'''scheme.3''' New parts BBa_I729005.]]
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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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===How Marimos are Made===
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===[[Chiba/Quorum_Sensing|Controlling Size]]===
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====Natural Marimos====
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[[Image:MLuxR-test.gif|frame|'''photo. 2''' Improved Receiver.]]
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Spherical marimos are formed through the growth of a core which is a fragment of ancestral marimos. So when you watch the cross section of the spherical marimo, you do not recognize a  core-shell structure in it. How about the most ancestral marimos?  It is expected that the most ancestral marimos had a core made of inorganic materials such as cray particles or sand solids.  
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#Improve Sender
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====Artificial Marimos====
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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
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Marimos are a popular "pet" in Japan. Well, you can't keep the National Tresure as a pet, so they house the artificial one instead. Although the mechanism of how the spherical marimo are made is not known in detail, the alternative method of making marimo have been invented from about 30 years ago by many Japanese scientist/botanist/marimoist.  
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#Improve Receiver
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#*[[Image:Chiba_check.png]] Inserted 2 mutations in LuxR which is known from the paper to increase activity.  
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#Localize AHL
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#*[[Image:Chiba_check.png]] Tested the GFP expression in constitutive aiiA generator receiver : Went too much.  
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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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#*[[Image:Chiba_nocheck.png]] AHL-induced inverter aiiA receiver. Not yet assembled.
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[[Chiba/Quorum_Sensing|more...]]
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[[Image:Chiba_marimohalf.png|frame|'''Fig3.''' The cutaway view of marimos.]]There are several ways to make artificial marimo, and you can buy the one made these ways at flowershops/ petshops in JPN. However, artificial marimo, or fake marimo, have clear difference from natural ones. When divided in the middle, the artificial marimos show no pattern or any sort of order. One can only see the fillament randomly tangled like a spaghetti. In contrast, natural marimo shows highly ordered structure of algae filaments; all filaments are lined up on an ordered way to form a radial pattern. In addition, the filament density of the core is very low (often nearly zero).
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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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==[[Chiba/Goal|Our Goal]]==
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Although we could not reach them, below we describe the goal that we set 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
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*A test to form Marimo
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*A test of size control
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[[Chiba/Goal| See Details]]
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[[Chiba/Marimo|more about marimos...]]
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===[[Chiba/Goal#In the Long Run|In the Long Run]]===
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See [[Chiba/Goal#In the Long Run|here]] : our brainstorming of the marimo future!
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References:
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[[Chiba/Goal|more...]]
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*阪井與志雄，マリモの科学，北海道大学図書刊行会, 1991 (Yoshio Sakai, Marimo no Kagaku（"The Science of Marimo"))
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*中沢信午，マリモはなぜ丸い　その生態と形態，中公新書，1989 (Shingo Nakazawa, Marimo wa naze marui ("Why marimos are spherical"))
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==Sponsers==
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<center>
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==Motivations==
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[[Image:Chiba_Knowledge.jpg]]　
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この内容だと、毬藻にできたら、何に応用できるか？という話なのだから、イントロ全体を変えないといけないと思う。細胞エンジニアリングの話だよね。んで、３次元細胞集合体をつくるということに頭をひねってみた。ちなみに細胞集合体で日本で有名なのは毬藻なんです、みたいな流れでないと。そうしたら、毬藻の生態の説明なんてごくわずかでも構わない（wikipediaにリンクだけっていうのも愛嬌で済むし）。by とよたろ<br>
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[[Image:Chiba_yakukensha.gif]]　
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[[Image:Chiba_Stratagene.gif]]　
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-->
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[[Image:Chiba_Greiner.gif]]<br><br>
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[[Image:Chiba-u.gif]]
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</center>

Latest revision as of 06:44, 27 October 2007

Chiba logo.png

Home
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

photo. 1 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.

Introduction: 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...

Project Design: How To Make Our Marimo

scheme. 1 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.

more...

Experiments Overview

Making Affinity Tags

scheme. 2 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

scheme.3 New parts BBa_I729005.
  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

photo. 2 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

Although we could not reach them, below we describe the goal that we set 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

See Details

In the Long Run

See here : our brainstorming of the marimo future!

more...

Sponsers

Chiba Knowledge.jpg　 Chiba yakukensha.gif　 Chiba Stratagene.gif　 Chiba Greiner.gif

Chiba-u.gif

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