Kyoto 2008 Cells as Power Source 50 years left - PowerPoint PPT Presentation

[Students] S. Takamori S. Hatakeyama S. Kazihata K. Sakuma G. Hayase M. Morita S. Kashida H. Kawahara [Advisers] M. Takinoue S-i. M. Nomura H. Saito Kyoto 2008

Cells as Power Source

50 years left http://commons.wikimedia.org/wiki/ Image:Titanic- bow_seen_from_MIR_I_submersible.jpeg

Cells as Power Source

i G . et Together i G ii. normous E E iii. achines M M GREAT s Cell as Power Source s

Size & Weight × 270000000 × 46000000000000000000000 270m 1µm (880ft) (1 × 10 -6 m) (p = 10 -12 ) 46Gg (G = 10 9 ) 1pg

Official Website Q&A Can the Titanic be raised? Sadly, even if the technology existed to raise it from the seabed, the wreck is far withstand liGing and transportaIon.

1 2 3 Gas Vesicle Flagella Binding Scheme to Produce Max of Total Power Max of Cell Number Gathering 1. binding To Solids Binding Peptides TBP To Non-solids PsBP Growth Chemotaxis 3. Flagella Quorum Sensing Che series Thresholds P Lux Lux proteins Positive FB +/- LuxI Metabolic Engineering Max of Power/Cell LuxR Motility of Cells swimming, swarming Flagella MotA, MotB gliding Proteorhodopsin twitching Che proteins CheA floating 2. Gas Vesicle Gas Vesicles CheZ Others, if any… Gvp series GvpA Ohters Different origins Cyanobacteria Lipids Ohters

1 2 3 Gas Vesicle Flagella Binding Outline • MoIvaIon and Target • Strategy: 3 STEPs 1. Binding 2. Gas 3. Flagella • Mechanical EsImaIon • Results & Conclusion

1 2 3 Gas Vesicle Flagella Binding Outline Strategy: 3 STEPs

1. SIcking to the Surface

2. Gas Vesicle Protein

3. Flagella RotaIon

1 2 3 Gas Vesicle Flagella Binding Design of Parts 1. Display of Material Binding Peptide BP Pconst. 2. Production of Gas Vesicle Protein (GVP) luxI luxR gvp Pconst. Plux Pconst. 3. Flagella Rotation Promoter cheZ RBS PompR Gene Terminator cph8 pcyA ho1 Pconst.

1. SIcking to the Surface

1 2 3 Binding Gas Vesicle Flagella Display of Material Binding PepIde Binding PepDde MoDfs Polystyrene: Binding Peptide FFSFFFPASAWGS Titanium: RKLPDAPGMHTW PS (542bp) Ti (524bp) Lpp signal sequence ＋ 1 st 9 a.a. 600 500 600 500 OmpA 46-159 a.a. PCR product

1 2 3 Binding Gas Vesicle Flagella Binding Assay Ti side PS side + IPTG - IPTG Ti side PS side TiBP PsBP Fluorescence microscopic images

2. Gas Vesicle Protein

2 1 3 Binding Flagella Gas Vesicle Gas Vesicle Protein(GVP) 2 µ m 0.5 µ m 28 gas vesicles can float a Electro microgragh single E.coli ， Gas vesicle in Prosthecomicrobium peuma4cum Reference: ANTHONY E. WALSBY Gas vesicle

2 1 3 Binding Flagella Gas Vesicle Problem of Gas Vesicle Too much expression of the Gvps leads to death. So the expression of Gvps should be controled. We use quorum sensing system!

2 1 3 Binding Flagella Gas Vesicle RegulaIon: Quorum Sensing Small cell populaIon Large cell populaIon AHL (quorum sensing molecule) Gas vesicles are produced

2 1 3 Binding Flagella Gas Vesicle Modeling (1) Moving (2) Dividing (3) AHL producing (4) GVP expression

2 1 3 Binding Flagella Gas Vesicle Results Quorum Sensing

2 1 3 Binding Flagella Gas Vesicle DifferenIaIon by AdaptaIon to Environment (DAE) <inside> High AHL Quorum sensing GVP dominant <surface> Low AHL Flagella dominant

3. Flagella RotaIon

1 2 3 Binding Gas Vesicle Flagella Can
really
bacteria
move
objects? YES YES


1 2 3 Binding Gas Vesicle Flagella Micro‐Bead Bacterial Flagella‐Based Propulsion and On/Off Mo4on Control of Microscale Objects Bahareh Behkam and Me4n SiC

1 2 3 Binding Gas Vesicle Flagella Bacteria Can Move a Bead Bacterial Flagella‐Based Propulsion and On/Off Mo4on Control of Microscale Objects Bahareh Behkam and Me4n SiC

1 2 3 Binding Gas Vesicle Flagella Two
Types
of
E.
Coli
Motion

 Tumbling Straight

1 2 3 Binding Gas Vesicle Flagella Che-Z
Makes
E.coli
Go
Straight 
 Wild Type Z Che‐Z Overexpressed

1 2 3 Binding Gas Vesicle Flagella Normal Our Idea Che-Z over-expressed

1 2 3 Binding Gas Vesicle Flagella Control of Che‐Z Expression cph8 ho1 pcyA Pconst. Che‐Z OmpR-controlled promoter

1 2 3 Binding Gas Vesicle Flagella Control of Che‐Z Expression cph8 ho1 pcyA Pconst. Che‐Z OmpR-controlled promoter

1 2 3 Gas Vesicle Flagella Binding Strategy: 3 STEPs

Mechanical EsImaIon Outline • MoIvaIon and Target • Strategy: 3 STEPs 1. Binding 2. Gas 3. Flagella • Mechanical EsImaIon • Result & Conclusion

Mechanical EsImaIon Mechanical EsImaIon Volume: 92,000 m 3 0.5 µm 3 0.25 m 2 Bottom Surface Area: 8,000 m 2

Mechanical EsImaIon Mechanical EsImaIon Total: 40,000 t

Mechanical EsImaIon Mechanical EsImaIon Total: 40,000 t Flagella ×(5.1×10 16 )

Mechanical EsImaIon Mechanical EsImaIon Total: 40,000 t Gas Vesicle ×(1.8×10 23 ) Flagella ×(5.1×10 16 )

Mechanical EsImaIon Mechanical EsImaIon Total: 40,000 t Gas Vesicle ×(1.8×10 23 ) Flagella ×(5.1×10 16 ) = 1.5×10 4 N

Mechanical EsImaIon Mechanical EsImaIon Total: 40,000 t Gas Vesicle × 80 ×(1.8×10 23 ) = 4.0×10 7 N 50% Flagella ×(5.1×10 16 ) = 1.5×10 4 N

Mechanical EsImaIon THE WIGHT OF THE TITANIC BECOME ZERO!

Mechanical EsImaIon Mechanical EsImaIon Gas Vesicle Gas Vesicle Flagella

Mechanical EsImaIon Mechanical EsImaIon Gas Vesicle Gas Vesicle Flagella

Result & Conclusion Outline • MoIvaIon and Target • Strategy: 3 STEPs 1. Binding 2. Gas 3. Flagella • Mechanical EsImaIon • Results & Conclusion

Result & Conclusion Making of Ti‐coated Boat Now coating…

Result & Conclusion Achievement Max of Total Power Max of Cell Number Gathering To Solids Binding Peptides TBP To Non-solids PsBP Growth Chemotaxis Quorum Sensing Che series Thresholds P Lux Lux proteins Positive FB +/- LuxI Metabolic Engineering Max of Power/Cell LuxR Motility of Cells swimming, swarming Flagella MotA, MotB gliding Proteorhodopsin twitching Che proteins CheA floating Gas Vesicles CheZ Others, if any… Gvp series GvpA Ohters Different origins Cyanobacteria Lipids Ohters

Result & Conclusion Achievement 1. binding 3. Flagella 2. Gas Vesicle To Be Continued…

Acknowledgments Technical Support Prof. Inoue, Dr. Siba, Prof. Shimamoto, Prof. Nakamura, and Prof. Yoshikawa, Dr. Fujita Financial Support Personal Supporters: K. Siba, N. Shimamoto, Y. Nakamura, S. Kato, T. Nakaya, K. Fujita, N. Yoshinaga, S. Kidoaki, A. Yamada, K. Tsumoto, H. Noguchi, Y. Yamasaki, S. Takagi, M. Ichikawa, D. Maezawa, Y. Kitahata, H. Mayama, T. Inoue, T. Hamada, T. Harada Special Thanks Laboratory of Gene Biodynamics at Kyoto University iGEM Chiba Team, Ms. Yoshida(Inoue Lab.)

+Osaka iGEM Kyoto