Marimo Bacteria Chiba iGEM 2007 Chiba University, Japan

iGEM Chiba Chiba University, Japan Hiroki Fukutomi Marina Gushiken Risa Kajiwara Mutsuko Kazama Yukihiro Takami Masahiro Tominaga

Motivation Chiba University, Japan Basu, S. et.al., Nature, 434, , (2005) Programmed shape formation Next Step: Sticking Gathering 3-D

Marimo Chiba University, Japan CoreGrow

Design

Devices Chiba University, Japan Sticker Communication Module Boundary Maker Affinity Tag Logic Circuit Switching threshold [AHL] Quorum Sensing

Chiba University, Japan Affinity Tag What needed to stick bacteria: Display & Affinity 1.Display Histidine-Tag On flagellar Co 2+ Histidine residence Co 2+ His-tag with Co 2+ 2.Affinity

Chiba University, Japan Affinity Tag (5-10 flagella per cell) Peptide insertable “variable” D3 domain Images from D3 fliC Histidine-tag 5’--3’

Chiba University, Japan Making Affinity Tag  Swarm confirmed  His-Tag displayed on flagella confirmed Swarm Non Swarm SDS-PAGE FliC Stained Histidine-Tag

Chiba University, Japan Affinity Check Method: Beads Adsorption Beads Co 2+ Drop out… Beads Adsorption

Chiba University, Japan Affinity Check Strain Method: Beads adsorption Exp. Affinity check Plasmids -FliC-His -No plasmid -JW1908(fliC) -GI826(fliC,motB) * motB deletion provides cell with the flagella completely assembled but not rotating.

Chiba University, Japan Devices Communication Module Boundary Maker Quorum Sensing Affinity Tag  Made FliC-His  Phenotype confirmed  Affinity confirmed Logic Circuit Switching threshold [AHL] Sticker

Chiba University, Japan Communication Module Purpose –To make the aggregation around the core How? –Quorum sensing pLux LuxI LuxR AHL LuxR

Chiba University, Japan Our Circuit Design pLux LuxR pTet pLac LuxR LuxI FliC-His pLux LuxR AHL Sender Receiver GFP

System Check Sender: BBa_S03623 Receiver:BBa_9002 pTet LuxI Chiba University, Japan Sender Receiver pLux LuxR pTet GFP Exp. Phenotype check

Chiba University, Japan Improvement Switching threshold [AHL] Power up! Switching threshold Super Sender Sensitive Receiver [AHL]

Super Sender AHL synthesizing pathway Construct Result Sell number pTet LuxIMetK Chiba University, Japan

Sensitive Receiver Collins et.al. Mol.Microbiol. 55, (2005 ) Collins’ mutant Construct Collins et.al. Mol.Microbiol. 55, (2005) Chiba University, Japan

Requirements Chiba University, Japan Communication Module Boundary Maker Quorum Sensing Affinity Tag  System check  Super sender  Sensitive receiver  Made FliC-His  Phenotype confirmed  Affinity confirmed Logic Circuit Switching threshold [AHL] Sticker

AHL Quencher Design Chiba University, Japan Switching threshold [AHL] Slope Express Flic Express aiiA Sensitive plux lacI aiiA plac cI plux Flic-his lacI [AHL] [Expression] Flic aiiA

AHL Quencher Design Chiba University, Japan Switching threshold [AHL] Slope Express Flic Express aiiA Sensitive plux lacI aiiA plac cI plux lacI Flic-his 1) No AHL [AHL] [Expression] Flic aiiA

AHL Quencher Design Chiba University, Japan Switching threshold [AHL] Slope Express Flic Express aiiA Sensitive plux lacI aiiA plac cI plux lacI flic-his 2) Few AHL aiiA [AHL] [Expression] Flic aiiA

AHL Quencher Design Chiba University, Japan Switching threshold [AHL] Slope Express Flic Express aiiA Sensitive plux lacI aiiA plac cI plux flic-his 3) High AHL lacI Flic-his [AHL] [Expression] Flic aiiA

aiiA Check Chiba University, Japan Switching threshold [AHL] Slope const-aiiA No fluorescence Normal receiver Fluorescence AHL pLux aiiAGFP plac Construct Result Experiment

Where we are: Chiba University, Japan Communication Module Boundary Maker Quorum Sensing Affinity Tag  System check  Super sender  Sensitive receiver  Made FliC-His  Phenotype confirmed  Affinity confirmed  Design circuit  AiiA check  Assemble circuit Logic Circuit Switching threshold [AHL] Sticker

Where we are: Chiba University, Japan Communication Module Boundary Maker Quorum Sensing Affinity Tag  System check  Super sender  Sensitive receiver  Made FliC-His  Phenotype confirmed  Affinity confirmed  Design circuit  AiiA check  Assemble circuit Logic Circuit Switching threshold [AHL] Sticker

Acknowledgements Chiba University, Japan