Chiba/Sandbox/Home2

From 2007.igem.org

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:

1. Make the sender core by sticking with the affinity tag.
2. Insert the sender core into the receiver culture.
3. The sender core produces AHL, which make the near receivers to generate the affinity tags and GFPs.
4. The affinity tagged receiver sticks with the central sender core. This will continue until the AHL cannnot reach the marimo boundary,
5. 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.
   • 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.

Making Marimo

Remaining Task(Work)

Our Goal

Future