1. Marimo Bacteria Chiba iGEM 2007 Chiba University, Japan

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

3. Motivation Chiba University, Japan Basu, S. et.al., Nature, 434, 1130-1134, (2005) Programmed shape formation Next Step: Sticking Gathering 3-D

4. Marimo Chiba University, Japan CoreGrow

5. Design

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

10. 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

11. Chiba University, Japan Affinity Tag (5-10 flagella per cell) Peptide insertable “variable” D3 domain Images from http://www.npn.jst.go.jp D3 fliC Histidine-tag 5’--3’

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

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

14. 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.

15. 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

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

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

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

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

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

21. Sensitive Receiver Collins et.al. Mol.Microbiol. 55, 712-723 (2005 ) Collins’ mutant Construct Collins et.al. Mol.Microbiol. 55, 712-723 (2005) Chiba University, Japan

22. 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

23. 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

24. 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

25. 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

26. 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

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

28. 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

29. 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

30. Acknowledgements Chiba University, Japan