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Chiba iGEM 2007

Chiba iGEM 2007. Marimo Bacteria. iGEM Chiba. Hiroki Fukutomi Marina Gushiken Risa Kajiwara. Mutsuko Kazama Yukihiro Takami Masahiro Tominaga. Next Step:. Sticking. Gathering. 3-D. Motivation. Programmed shape formation. Basu, S. et.al., Nature , 434, 1130-1134, (2005). Marimo.

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Chiba iGEM 2007

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  1. Chiba iGEM 2007 Marimo Bacteria Chiba University, Japan

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

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

  4. Marimo Core Grow Chiba University, Japan

  5. Design

  6. Design

  7. Design

  8. Design

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

  10. Affinity Tag 2.Affinity 1.Display His-tag with Co2+ Histidine-Tag On flagellar Histidine residence Co2+ Co2+ What needed to stick bacteria: Display & Affinity Chiba University, Japan

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

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

  13. Affinity Check Method: Beads Adsorption Beads Adsorption Beads Co2+ Co2+ Drop out… Chiba University, Japan

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

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

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

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

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

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

  20. Super Sender AHL synthesizing pathway Result Construct LuxI MetK pTet Sellnumber Chiba University, Japan

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

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

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

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

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

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

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

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

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

  30. Acknowledgements Chiba University, Japan

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