chiba igem 2007
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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, 1130-1134, (2005)
Programmed shape formationNext Step:
Sticking
Gathering
3-D
Marimo
Chiba University, Japan
Core Grow
Design
Design
Design
Design
Devices
Chiba University, Japan
StickerCommunication
Module Boundary Maker
Affinity Tag Logic Circuit
Switchingthreshold
[AH
L]
Quorum Sensing
Chiba University, Japan
Affinity TagWhat needed to stick bacteria:
Display & Affinity
1.DisplayHistidine-Tag On flagellar
Co2+
Histidine residence
Co2+
His-tag with Co2+
2.Affinity
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’
Chiba University, Japan
Making Affinity Tag
Swarm confirmedHis-Tag displayed on flagella confirmed
Swarm Non Swarm SDS-PAGE
FliC
Stained Histidine-Tag
Chiba University, Japan
Affinity CheckMethod: Beads Adsorption
Beads
Co2+ Co2+
Drop out…
BeadsAdsorption
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 flagellacompletely assembled but not rotating.
Chiba University, Japan
Devices
CommunicationModule Boundary Maker
Quorum SensingAffinity Tag
Made FliC-HisPhenotype confirmedAffinity confirmed
Logic Circuit
Switchingthreshold
[AH
L]
Sticker
Chiba University, Japan
Communication Module
• Purpose– To make the aggregation around the core
• How?– Quorum sensing
pLux
LuxILuxR
AHLLuxR
Chiba University, Japan
Our Circuit Design
pLuxLuxR
pTet
pTet pLac
LuxR
LuxI FliC-His
FliC-HispLux
LuxR
AHLAHL
AHLAHL
AHL
Sender
Receiver
GFP
System Check
Sender: BBa_S03623
Receiver:BBa_9002
pTetLuxI
Chiba University, Japan
Sender
Receiver
pLuxLuxR
pTetGFP
Exp. Phenotype check
Chiba University, Japan
Improvement
Switchingthreshold
[AH
L]
Power up!
Switchingthreshold
Super Sender Sensitive Receiver
[AH
L]
Super SenderAHL synthesizing pathway
Construct
Result
Sell number
pTetLuxI MetK
Chiba University, Japan
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
Requirements
Chiba University, Japan
CommunicationModule Boundary Maker
Quorum SensingAffinity Tag
System checkSuper senderSensitive receiver
Made FliC-HisPhenotype confirmedAffinity confirmed
Logic Circuit
Switchingthreshold
[AH
L]
Sticker
AHL Quencher Design
Chiba University, Japan
Switchingthreshold
[AH
L]
Slope
Express Flic
Express aiiA
Sensitive pluxlacI
aiiAplac
cI plux Flic-hislacI
[AH
L]
[Expression]
FlicaiiA
AHL Quencher Design
Chiba University, Japan
Switchingthreshold
[AH
L]
Slope
Express Flic
Express aiiA
Sensitive pluxlacI
aiiAplac
cI pluxlacI Flic-his1) No AHL
[AH
L]
[Expression]
FlicaiiA
AHL Quencher Design
Chiba University, Japan
Switchingthreshold
[AH
L]
Slope
Express Flic
Express aiiA
Sensitive pluxlacI
aiiAplac
cI pluxlacI flic-his2) Few AHL
aiiA
[AH
L]
[Expression]
FlicaiiA
AHL Quencher Design
Chiba University, Japan
Switchingthreshold
[AH
L]
Slope
Express Flic
Express aiiA
Sensitive pluxlacI
aiiAplac
cI plux flic-his3) High AHL lacI
Flic-his
[AH
L]
[Expression]
FlicaiiA
aiiA Check
Chiba University, Japan
Switchingthreshold
[AH
L]
Slope
const-aiiANo fluorescence
Normal receiverFluorescence
AHL
pLuxaiiA GFP
plac
Construct
Result
Experiment
Where we are:
Chiba University, Japan
CommunicationModule Boundary Maker
Quorum SensingAffinity Tag
System checkSuper senderSensitive receiver
Made FliC-HisPhenotype confirmedAffinity confirmed
Design circuitAiiA checkAssemble circuit
Logic Circuit
Switchingthreshold
[AH
L]
Sticker
Where we are:
Chiba University, Japan
CommunicationModule Boundary Maker
Quorum SensingAffinity Tag
System checkSuper senderSensitive receiver
Made FliC-HisPhenotype confirmedAffinity confirmed
Design circuitAiiA checkAssemble circuit
Logic Circuit
Switchingthreshold
[AH
L]
Sticker
Acknowledgements
Chiba University, Japan