Chiba/Sandbox/Home2
From 2007.igem.org
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Introduction | Project Design ( 1.Affinity Tag | 2.Communication Module | 3.Size Control ) | Making Marimos | Our Goal |
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.
(more...)
How To Make Our Marimo
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:
Experiments Overview
Making Affinity Tags
Fig. The short peptide with six histidine (“His-Tag”) was inserted into the fliC D3 domain.
- 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
Making Communication Module
- Making Receivers
- AHL > GFP generator (constitutive aiiA) : BBa_T729006
- AHL > GFP & aiiA generator : BBa_I729005
- Sensitive AHL > GFP generator : BBa_I729004
inverter-aiiA
- Making Senders
- MetK Sender ( could not deposit to registry )
Controlling Size
- Improve Sender
- Overexpressed the metK (the AHL precursur synthesis enzyme) in the hope to increase AHL synthesis. (Turned out not to work)
- Improve Receiver
- Inserted 2 mutations in LuxR which is known from the paper to increase activity.
- 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.
