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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

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.
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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:

• 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

1. Make the affinity tag by inserting the his-tag into the flagellar fillament.
   • Sequence confirmed
   • Swarm confirmed
   • Flagella strained with anti-flagella antibody
   • Phenotype confirmed
   • Affnity confirmed

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Making Communication Module

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

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Controlling Size

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

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Making Marimo

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

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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!

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