chiba igem 2007

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