Chiba/Sandbox/Home2

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Our marimo bacteria requres as follows:
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===How To Make Our Marimo===
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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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'''1.Affinity Tag.'''
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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 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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'''2.Communication Module.'''
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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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'''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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こまかすぎか．
 
==Experiments Overview==
==Experiments Overview==

Revision as of 04:21, 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 | iGEM Chiba Website | メンバ連絡簿

Project Overview

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?) Motivation.
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How To Make Our Marimo

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:

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

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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 )

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Controlling Size

  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.

Making Marimo

Remaining Task(Work)

Our Goal

Future

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